Anti-trem2 antibodies and methods of use thereof

ABSTRACT

The invention is generally directed to methods and compositions that include antibodies, e.g., monoclonal, chimeric, humanized antibodies, antibody fragments, etc., that specifically bind a TREM2 protein, e.g., a mammalian TREM2 and/or human TREM2. The methods provided herein find use in preventing, reducing risk, or treating an individual having dementia, frontotemporal dementia, Alzheimer&#39;s disease, Nasu-Hakola disease, or multiple sclerosis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/035,336, filed Aug. 8, 2014, U.S. Provisional Application No.62/135,110, filed Mar. 18, 2015, and U.S. Provisional Application No.62/135,122, filed Mar. 18, 2015, each of which is hereby incorporated byreference in its entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name:735022000440SEQLISTING.TXT, date recorded: Aug. 7, 2015, size: 240 KB).

FIELD OF THE INVENTION

This invention relates to anti-TREM2 and anti-DAP12 antibodies andtherapeutic uses of such antibodies.

BACKGROUND OF THE INVENTION

Triggering receptor expressed on myeloid cells-2 (TREM2) is animmunoglobulin-like receptor that is expressed primarily on myeloidlineage cells, such as macrophages, dendritic cells, monocytes,Langerhans cells of skin, Kupffer cells, osteoclasts, and microglia; andis required for modulation (e.g., suppression) of Toll-like receptor(TLR) signaling, the modulation of inflammatory cytokines, as well asfor normal osteoclast development. TREM2 was discovered as a member ofthe TREM transmembrane glycoproteins, which belong to the singleimmunoglobulin variable (IgV) domain receptor family. The genes encodinghuman and mouse TREMs map to human chromosome 6p21.1 and mousechromosome 17C3, respectively. The TREM cluster includes genes encodingTREM1, TREM2, TREM4, and TREM5, as well as the TREM-like genes in bothhuman and mouse. Additionally TREM3 and plasmocytoid dendritic cell(pDC)-TREM were identified in mouse. The TREM-like genes, TREML1 andTREML2 in humans, and Treml1 and Treml2 in mouse, encode TLT-1 and TLT-2respectively. The two best characterized of these receptors, TREM1 andTREM2, display some sequence homology with other members of the Ig-SFsuch as activating NK cells receptors (20% identity with NKp44) and actthrough association with a DAP12-mediated pathway for signaling.

TREM2 was originally cloned as a cDNA encoding a TREM1 homologue(Bouchon, A et al., J Exp Med, 2001. 194(8): p. 1111-22). This receptoris a glycoprotein of about 40 kDa, which is reduced to 26 kDa afterN-deglycosylation. The TREM2 gene encodes a 230 amino acid-lengthprotein that includes an extracellular domain, a transmembrane regionand a short cytoplasmic tail. The extracellular region, encoded by exon2, is composed of a single type V Ig-SF domain, containing threepotential N-glycosylation sites. The putative transmembrane regioncontains a charged lysine residue. The cytoplasmic tail of TREM2 lackssignaling motifs and is thought to signal through the signaling adaptormolecule DAP12/TRYROBP.

The signaling adaptor molecule DAP12 is expressed as a homodimer at thesurface of a variety of cells participating in innate immune response,including microglia, macrophages, granulocytes, NK cells, and dendriticcells (DC). DAP12 is a member of the type I transmembrane adapterprotein family on the basis of homology with the human T-cell receptor(TCR)-associated CD3 chains and the Fc receptor (FcR) γ-chain (Turnbull,I R and Colonna, M, Nat Rev Immunol, 2007. 7(2): p. 155-61). Theseproteins share many structural and functional characteristics, includingone or more ITAM motifs in their cytoplasmic domain, charged acidicresidue in transmembrane region (critical for interaction with itspartner chain) and the ability to recruit Src homology domain-2(SH2)-containing proteins following tyrosine phosphorylation. The ITAMmotif mediates signal propagation by activation of the ZAP70 or Syktyrosine kinase. Both kinases phosphorylate several substrates, therebyfacilitating the formation of a signaling complex leading to cellularactivation. Interestingly, some B-cells and T-cells also express DAP12under inflammatory conditions. In humans, subsets of CD4⁺CD28⁻ T-cells,αβTCR⁺CD4⁺ T-cells, and CD8⁺ T-cells expressing this protein have beendescribed in patients suffering from chronic inflammatory diseases, inthe context of autoimmune T cells (Schleinitz, N. et al., PLoS ONE, 4(2009), p. e6264). In view of the significant level of DAP12 expressionin mouse peritoneal macrophages, this protein is believed to beexpressed in other macrophage-related cells, such as osteoclasts in thebone marrow, Kupffer cells in the liver, alveolar macrophages of thelung, Langerhans cells of skin, and microglial cells in the brain(Takaki, R et al., Immunol Rev, 2006. 214: p. 118-29).

TREM2 has been identified as expressed on the surface of humanmonocyte-derived dendritic cells and as an mRNA transcript in the mousemacrophage cell line RAW264 (Bouchon, A et al., J Exp Med, 2001. 194(8):p. 1111-22). Human TREM2 was the first DAP12-associated receptordescribed on the surface of DCs. Studies have demonstrated that TREM2cell surface expression is reduced in DAP12-deficient bonemarrow-derived dendritic cells (BMDCs) and in DAP12-deficientmacrophages, as compared to wild-type cells (Ito, H and Hamerman, J A,Eur J Immunol. 42(1): p. 176-85; Hamerman, J A et al., J Immunol, 2006.177(4): p. 2051-5; and Hamerman, J A et al., Nat Immunol, 2005. 6(6): p.579-86). This indicates that formation of the TREM2/DAP12 complex isneeded for maximal TREM2 surface expression.

Recent studies have also shown cell-surface expression of TREM2 onmacrophages infiltrating tissue from the circulation, as well as onmacrophages activated by IL-4 or IL-13 (Turnbull, I R et al., J Immunol,2006. 177(6): p. 3520-4). However, TREM2 expression was not always foundin other cell populations, such as tissue-resident macrophages,circulating monocytes, or the corresponding progenitor cells in the bonemarrow, suggesting that TREM2 expression is not induced centrally, butlocally during tissue infiltration or by cytokine-mediated activation.Moreover, it has also been observed that IFN-γ and LPS reduce orotherwise abrogate TREM2 expression. Further, it has been recentlyreported that TREM2 is highly expressed on microglia and infiltratingmacrophages in the central nervous system during experimental autoimmuneencephalomyelitis or Alzheimer's disease (Piccio, L et al., Eur JImmunol, 2007. 37(5): p. 1290-301; and Wang Y, Cell. 2015 Mar. 12;160(6):1061-71).

It has been shown that TREM2 signals through DAP12. Downstream thisleads to activation of the Syk/Zap70 tyrosine kinase family, PI3K, andother intracellular signals. On myeloid cells, TLR signals are importantfor activation, such as with infection response, but also play a keyrole in the pathological inflammatory response, such as with macrophagesand dendritic cells (Hamerman, J A et al., (2006) J Immunol 177:2051-2055; Ito, H et al., Eur J Immunol 42: 176-185; Neumann, H et al.,(2007) J Neuroimmunol 184: 92-99; Takahashi, K et al., (2005) J Exp Med201: 647-657; and Takahashi, K et al., (2007) PLoS Med 4: e124).Deficiency of either TREM2 or DAP12 is thought to lead to increasedpro-inflammatory signaling. The impact of TREM2-deficiency in vitro hasbeen shown in the context of stimulation with typical TLR ligands, suchas LPS, CpG DNA, and Zymosan. TREM-2-deficient dendritic cells showincreased release of IL-12p70, TNF, IL-6, and IL-10 in the presence, butnot in the absence of stimulation.

Several recent studies have explored the intracellular signaling eventsinduced by the activation of the TREM2/DAP12 pathway. For example, TREM2is thought to activate signaling pathways involved in cell survival(e.g., protein kinase B-Akt), cell activation and differentiation (e.g.,Syk, Erk1/2, PLC-γ, elc.), and in the control of the actin cytoskeleton(e.g., Syk, Vav, etc.) (Peng, Q et al., Sci Signal. 3(122): p. ra38; andWhittaker, G C et al., J Biol Chem. 285(5): p. 2976-85). After ligationof TREM2, the ITAM tyrosines in DAP12 are phosphorylated by SRC-familykinases leading to the recruitment and activation of the Syk kinaseand/or ZAP70 kinase. In the mouse, Syk may be the predominant kinaseinvolved, whereas in humans both Syk and ZAP70 appear to coupleefficiently with such ITAM-containing subunits, binding them throughtheir tandem SH2 domains.

Studies on TREM2 signaling have shown that, like TREM1, TREM2-mediatedsignaling through DAP12 also leads to an increase in intracellularcalcium ion levels and ERK1/2 phosphorylation of ERK1/2 (Bouchon, A etal., J Exp Med, 2001. 194(8): p. 1111-22; and Sharif, O and Knapp, S,Immunobiology, 2008. 213(9-10): p. 701-13). Importantly, TREM2 receptorligation does not induce the degradation of IkB-a and the subsequentnuclear translocation of NF-kB, which points to a possible differencebetween TREM2 and TREM1 signaling (Bouchon, A et al., J Exp Med, 2001.194(8): p. 1111-22). Receptor cross-linking of TREM2 on immaturedendritic cells triggers the up-regulation of molecules involved inT-cell co-stimulation, such as CD86, CD40, and MHC class II, as well asthe up-regulation of the chemokine receptor CCR7 (Bouchon, A et al., JExp Med, 2001. 194(8): p. 1111-22). TREM2 is also expressed onmicroglia, where receptor cross-linking results in an increase in ERK1/2phosphorylation and CCR7, but not an increase in CD86 or MHC class IIexpression, suggesting possible cell type-specific differences in TREM2signaling. Additionally, over-expression of TREM2 in myeloid cellsresulted in an increase in phagocytosis of degenerated myelin(Takahashi, K et al., PLoS Med, 2007. 4(4): p. e124; and Neumann, H andTakahashi, K, J Neuroimmunol, 2007. 184(1-2): p. 92-9).

It has also been shown that bone marrow-derived macrophages (BMDM) thathave been silenced for TREM2 using shRNAi display increased secretion ofTNF in response to the TLR2/6 ligand zymosan and the TLR9 ligand CpG, ascompared to control BMDM cells that were treated with a non-specificshRNAi, indicating that TREM2 negatively regulates cytokine synthesis inmacrophages (Ito, H and Hamerman, J A, Eur J Immunol. 42(1): p. 176-85;Hamerman, J A et al., J Immunol, 2006. 177(4): p. 2051-5; and Hamerman,J A et al., Nat Immunol, 2005. 6(6): p. 579-86). These results have beenconfirmed using BMDM cells from TREM2 knockout mice, and have furthershown that levels of TNF and IL-6 were also higher in TREM2^(−/−) BMDMcells in response to LPS, as compared to wild-type BMDM cells (Turnbull,I R, et al., J Immunol, 2006. 177(6): p. 3520-4; and Turnbull, I R andColonna, M, Nat Rev Immunol, 2007. 7(2): p. 155-61). Additionally, TREM2overexpression in microglia has been demonstrated to lead to a decreasein TNF and inducible nitric oxide (iNOS) mRNA after culture of thesecells with apoptotic neurons, whereas TREM2 knockdown resulted in amodest increase in TNF and iNOS mRNA levels. This indicates that, incontrast to TREM1, which is a positive regulator of cytokine synthesis,TREM2 is a negative regulator of cytokine synthesis. This effect ofTREM2 on inflammation may be independent of the type of macrophage as itoccurs in both microglia and BMDM cells.

It has also been shown that in resident myeloid cells of the centralnervous system, activation of microglia can lead to inflammation(Neumann, H et al., (2007) J Neuroimmunol 184: 92-99; Takahashi, K etal., (2005) J Exp Med 201: 647-657; Takahashi, K et al., (2007) PLoS Med4: e124; and Hsieh, C L et al., (2009) J Neurochem 109: 1144-1156).Moreover, microglia activation has also been implicated infrontotemporal dementia (FTD), Alzheimer's disease, Parkinson's disease,stroke/ischemic brain injury, and multiple sclerosis. Whereas reducedTREM2 activation leads to increases in certain activation andinflammation markers, such as NOS2 gene transcription in myeloid cells,increased TREM2 activation leads to reduced NOS2 transcription. It isthought that dying neurons express an endogenous ligand for TREM2. HSP60has been implicated as a ligand of TREM2 on neuroblastoma cells(Stefani, L et al., (2009) Neurochem 110: 284-294). TREM2over-expression also leads to increased phagocytosis of dying neurons bymicroglia, and similarly increases phagocytosis by other myeloid lineagecells.

In humans, the complete absence of TREM2 has been shown to causeNasu-Hakola disease, a rare neurodegenerative disease with late-onsetdementia, demyelination, and cerebral atrophy (Paloneva, J et al.,(2002) Am J Hum Genet 71: 656-662; and Paloneva, J et al., (2003) J ExpMed 198: 669-675). Nasu-Hakola disease can also be caused byDAP12-deficiency.

TREM2 gene expression has also been shown to be increased in APP23transgenic mice, an Alzheimer's disease model in which the mice expressa mutant form of the amyloid precursor protein that is associated withfamilial Alzheimer's disease (Melchior, B et al., ASN Neuro 2: e00037).Uptake of Amyloid 1-42 has also been shown to be increased in BV-2microglial cell lines that overexpress TREM2.

TREM2 has further been shown to be upregulated in the EAE mouse model ofmultiple sclerosis (Neumann, H et al., (2007) J Neuroimmunol 184: 92-99;Takahashi, K et al., (2005) J Exp Med 201: 647-657; and Takahashi, K etal., (2007) PLoS Med 4: e124). The transduction of bone marrow-derivedmyeloid precursor cells (BM-DC) in vitro with TREM2 leads to increasedphagocytosis of degenerated myelin. In response to LPS, these cells showincreased IL-10 and decreased IL-1β. Intravenous transplantation ofmyeloid cells overexpressing TREM2 can suppress EAE in vivo.

Further, exome sequencing of individuals with frontotemporal dementia(FTD) presentation has identified homozygous mutations in TREM2(Guerreiro, R J et al., JAMA Neurol 70: 78-84; and Guerreiro, R J etal., Arch Neurol: 1-7). Some of these mutations lead to truncation andlikely loss-of-function of TREM2. These same TREM2 mutations can alsocause Naku-Hakula disease in some individuals. Imaging analysis incertain individuals with TREM2 homozygous mutations has also shownevidence of demyelination.

Heterozygous mutations in TREM2, which are the same as the mutation thatcause Naku-Hakula and FTD, also increase the risk of Alzheimer's disease(Guerreiro, R et al., N Engl J Med 368: 117-127; Jonsson, T et al., NEngl J Med 368: 107-116; and Neumann, H et al., N Engl J Med 368:182-184). Although these TREM2 mutations are rarer than the known riskvariants of Alzheimer's disease (e.g., APOE4), the effect of carryingthese mutations is just as serious; around a 3 fold increase in the riskof developing Alzheimer's disease. Moreover, even individuals withoutAlzheimer's disease who carry a heterozygous TREM2 mutation show worsecognition as compared to individuals with two normal TREM2 alleles.Further, it has been shown that the R47H variant of TREM2 (arginine tohistidine amino acid substitution at position 47 of TREM2), which ismost common TREM2 mutation (up to 1 in 200 individuals) is locatedwithin the immunoglobulin domain of TREM2, and may thus alter ligandbinding (Wang Y, Cell. 2015 Mar. 12; 160(6):1061-71).

In addition an integrative network-based approach to rank-orderedorganized structure of molecular networks of gene expression forrelevance to late onset developing Alzheimer's disease (LOAD) identifiedTYROBP/DAP12 as the signaling molecule for TREM2 as a key regulator ofthe immune/microglia gene modules that is associated with LOAD. TYROBPwas found to be the causal regulator of the highest scoringimmune/microglia module as rank-ordered based on the number of othergenes that TREM2 regulated and the magnitude of loss of regulation, aswell as differential expression in LOAD brains. TYROBP was significantlyupregulated in LOAD brains and there was a progression of TYROBPexpression changes across mild cognitive impairment (MCI), which oftenprecedes LOAD (Zhang et al., (2013) Cell 153, 707-720). Targeting suchcausal networks in ways that restore them to a normal state may be a wayto treat disease.

Accordingly, there is a need for antibodies that specifically bind TREM2and/or its signaling adapter molecule DAP12/TRYROBP on a cell surfaceand that modulate (e.g., activate or inhibit) one or more TREM2 and/orDAP12 activities in order to treat one or more diseases, disorders, andconditions associated with decreased TREM2 and/or DAP12 activity, aswell as conditions associated with undesired TREM2 and/or DAP12activity.

Moreover, the tumor microenvironment is composed of a heterogeneousimmune infiltrate, which include T lymphocytes, macrophages and cells ofmyeloid/granulocytic lineage. Therapeutic approaches that modulatespecific subsets of immune cells are changing the standard of care.“Checkpoint blocking” antibodies targeting immune-modulatory moleculesexpressed on T cells (such as CTLA-4 and PD-1) have demonstratedclinical activity across a variety of tumor types (Naidoo-et al., (2014)British Journal of Cancer 111, 2214-2219).

Cancer immune-therapy targeting tumor-associated macrophages (e.g.,M2-type macrophages) is an intense area of research. The presence ofM2-macrophages in tumors is associated with poor prognosis. Accordingly,there is a need for antibodies that specifically bind TREM2 and/or DAP12and (e.g., activate or inhibit) one or more TREM2 and/or DAP12activities in tumor-associated immune cells, such as macrophages,dendritic cells, myeloid/granulocytic cells, T cells, and monocytes.

All references cited herein, including patent applications andpublications, are hereby incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

The invention is generally directed to methods and compositions thatinclude antibodies, e.g., monoclonal antibodies, chimeric antibodies,bispecific antibodies, humanized antibodies, antibody fragments, etc.,that specifically bind a TREM2 protein and/or its signaling adaptormolecule DAP12, e.g., a mammalian TREM2, a human TREM2, a mammalianDAP12, or a human DAP12, including wild-type proteins and naturallyoccurring variants thereof. The antibodies of the present disclosure mayinclude agonist antibodies, inert antibodies, and/or antagonistantibodies. The methods provided herein find use in preventing, reducingrisk, or treating an individual having dementia, frontotemporaldementia, Alzheimer's disease, Nasu-Hakola disease, or multiplesclerosis; in inducing or promoting innate immune cell survival in anindividual in need thereof, and/or in decreasing innate immune cellsurvival in an individual in need thereof.

Certain aspect of the present disclosure relate to different classes ofanti-TREM2 antibodies. In some embodiments, anti-TREM2 antibodies areagonist antibodies that bind to TREM2 and activate, induce, promote,stimulate, or otherwise increase one or more TREM2 activities, survivalof one or more innate immune cells, and/or expression of IL-6. In someembodiments, agonist anti-TREM2 antibodies of the present disclosurecompete with TREM2 ligands for binding to TREM2 expressed on a cellsurface. In some embodiments, agonist anti-TREM2 antibodies of thepresent disclosure do not compete with TREM2 ligands for binding toTREM2 expressed on a cell surface. In some embodiments, anti-TREM2antibodies are inert or antagonist antibodies that bind to TREM2 anddecrease, inhibit, or otherwise reduce one or more TREM2 activitiesand/or survival of one or more innate immune cells. In some embodiments,inert or antagonist anti-TREM2 antibodies of the present disclosureblock or otherwise inhibit ligand binding to TREM2 expressed on a cellsurface.

Other aspects of the present disclosure relate to an isolated agonistantibody that binds to a TREM2 protein, a DAP12 protein, or both,wherein the antibody induces one or more TREM2 activities, DAP12activities, or both.

In certain embodiments that may be combined with any of the precedingembodiments, the TREM2 protein, the DAP12 protein, or both is amammalian protein or a human protein. In certain embodiments that may becombined with any of the preceding embodiments, the TREM2 protein, theDAP12 protein, or both is a wild-type protein. In certain embodimentsthat may be combined with any of the preceding embodiments, the TREM2protein, the DAP12 protein, or both is a naturally occurring variant. Incertain embodiments that may be combined with any of the precedingembodiments, the TREM2 protein, the DAP12 protein, or both is expressedon human dendritic cells, human macrophages, human monocytes, humanosteoclasts, human Langerhans cells of skin, human Kupffer cells, and/orhuman microglia. In certain embodiments that may be combined with any ofthe preceding embodiments, the isolated antibody induces or retainsTREM2 clustering, DAP12 clustering, or both on a cell surface. Incertain embodiments that may be combined with any of the precedingembodiments, the one or more TREM2 activities comprise TREM2 binding toDAP12. In certain embodiments that may be combined with any of thepreceding embodiments, the one or more DAP12 activities comprise DAP12binding to TREM2. In certain embodiments that may be combined with anyof the preceding embodiments, the one or more TREM2 activities, DAP12activities, or both comprise DAP12 phosphorylation, TREM2phosphorylation, or both. In certain embodiments that may be combinedwith any of the preceding embodiments, DAP12 phosphorylation, TREM2phosphorylation, or both is induced by one or more SRC family tyrosinekinases. In certain embodiments that may be combined with any of thepreceding embodiments, the one or more SRC family tyrosine kinasescomprise a Syk kinase. In certain embodiments that may be combined withany of the preceding embodiments, the one or more TREM2 activities,DAP12 activities, or both comprise PI3K activation. In certainembodiments that may be combined with any of the preceding embodiments,the one or more TREM2 activities, DAP12 activities, or both compriseincreased expression of one or more anti-inflammatory cytokines. Incertain embodiments that may be combined with any of the precedingembodiments, the one or more TREM2 activities, DAP12 activities, or bothcomprise increased expression of one or more anti-inflammatory mediators(e.g., cytokines) selected from the group consisting of IL-12p70, IL-6,and IL-10. In certain embodiments that may be combined with any of thepreceding embodiments, the increased expression occurs in one or morecells selected from the group consisting of macrophages, dendriticcells, monocytes, osteoclasts, Langerhans cells of skin, Kupffer cells,and microglial cells. In certain embodiments that may be combined withany of the preceding embodiments, the one or more TREM2 activities,DAP12 activities, or both comprise reduced expression of one or morepro-inflammatory cytokines. In certain embodiments that may be combinedwith any of the preceding embodiments, the one or more TREM2 activities,DAP12 activities, or both comprise reduced expression of one or morepro-inflammatory mediators selected from the group consisting of IFN-a4,IFN-b, IL-6, IL-12 p70, IL-1β, TNF, TNF-α, IL-10, IL-8, CRP, TGF-betamembers of the chemokine protein families, IL-20 family members, IL-33,LIF, IFN-gamma, OSM, CNTF, TGF-beta, GM-CSF, IL-11, IL-12, IL-17, IL-18,and CRP. In certain embodiments that may be combined with any of thepreceding embodiments, the one or more TREM2 activities, DAP12activities, or both comprise reduced expression of TNF-α, IL-6, or both.In certain embodiments that may be combined with any of the precedingembodiments, the reduced expression of the one or more pro-inflammatorymediators occurs in one or more cells selected from the group consistingof macrophages, dendritic cells, monocytes, osteoclasts, Langerhanscells of skin, Kupffer cells, and microglial cells. In certainembodiments that may be combined with any of the preceding embodiments,the one or more TREM2 activities, DAP12 activities, or both compriseextracellular signal-regulated kinase (ERK) phosphorylation. In certainembodiments that may be combined with any of the preceding embodiments,the one or more TREM2 activities, DAP12 activities, or both compriseincreased expression of C-C chemokine receptor 7 (CCR7). In certainembodiments that may be combined with any of the preceding embodiments,the one or more TREM2 activities, DAP12 activities, or both compriseinduction of microglial cell chemotaxis toward CCL19 and CCL21expressing cells. In certain embodiments that may be combined with anyof the preceding embodiments, the one or more TREM2 activities, DAP12activities, or both comprise an enhancement, normalization, or both ofthe ability of bone marrow-derived dendritic cells to induceantigen-specific T-cell proliferation. In certain embodiments that maybe combined with any of the preceding embodiments, the one or more TREM2activities, DAP12 activities, or both comprise induction of osteoclastproduction, increased rate of osteoclastogenesis, or both. In certainembodiments that may be combined with any of the preceding embodiments,the one or more TREM2 activities, DAP12 activities, or both compriseincreasing the survival of macrophages, microglial cells, or both. Incertain embodiments that may be combined with any of the precedingembodiments, the one or more TREM2 activities, DAP12 activities, or bothcomprise increasing the function of macrophages, microglial cells,dendritic cells, monocytes, osteoclasts, Langerhans cells of skin,and/or Kupffer cells. In certain embodiments that may be combined withany of the preceding embodiments, the macrophages are M1 macrophagesand/or microglia, M2 macrophages and/or microglia, or both. In certainembodiments that may be combined with any of the preceding embodiments,the M1 macrophages and/or microglia are activated M1 macrophages and/ormicroglia. In certain embodiments that may be combined with any of thepreceding embodiments, the one or more TREM2 activities, DAP12activities, or both comprise induction of one or more types of clearanceselected from the group consisting of apoptotic neuron clearance, nervetissue debris clearance, non-nerve tissue debris clearance, bacteria orother foreign body clearance, disease-causing protein clearance,disease-causing peptide clearance, and disease-causing nucleic acidclearance. In certain embodiments that may be combined with any of thepreceding embodiments, the one or more TREM2 activities, DAP12activities, or both comprise induction of phagocytosis of one or more ofapoptotic neurons, nerve tissue debris, non-nerve tissue debris,bacteria, other foreign bodies, disease-causing proteins,disease-causing peptides, or disease-causing nucleic acid. In certainembodiments that may be combined with any of the preceding embodiments,the disease-causing protein is selected from the group consisting ofamyloid beta or fragments thereof, Tau, IAPP, alpha-synuclein, TDP-43,FUS protein, prion protein, PrPSc, huntingtin, calcitonin, superoxidedismutase, ataxin, Lewy body, atrial natriuretic factor, islet amyloidpolypeptide, insulin, apolipoprotein AI, serum amyloid A, medin,prolactin, transthyretin, lysozyme, beta 2 microglobulin, gelsolin,keratoepithelin, cystatin, immunoglobulin light chain AL, S-IBM protein,Repeat-associated non-ATG (RAN) translation products, DiPeptide repeat(DPR) peptides, glycine-alanine (GA) repeat peptides, glycine-proline(GP) repeat peptides, glycine-arginine (GR) repeat peptides,proline-alanine (PA) repeat peptides, and proline-arginine (PR) repeatpeptides. In certain embodiments that may be combined with any of thepreceding embodiments, the disease-causing nucleic acid is antisenseGGCCCC (G2C4) repeat-expansion RNA. In certain embodiments that may becombined with any of the preceding embodiments, the one or more TREM2activities, DAP12 activities, or both comprise normalization ofdisrupted TREM2/DAP12-dependent gene expression. In certain embodimentsthat may be combined with any of the preceding embodiments, the one ormore TREM2 activities, DAP12 activities, or both comprise recruitment ofSyk, ZAP70, or both to a DAP12/TREM2 complex. In certain embodimentsthat may be combined with any of the preceding embodiments, the one ormore TREM2 activities, DAP12 activities, or both comprise Sykphosphorylation. In certain embodiments that may be combined with any ofthe preceding embodiments, the one or more TREM2 activities, DAP12activities, or both comprise increased expression of CD83 and/or CD86 ondendritic cells, macrophages, and/or monocytes. In certain embodimentsthat may be combined with any of the preceding embodiments, the one ormore TREM2 activities, DAP12 activities, or both comprise reducedsecretion of one or more inflammatory cytokines. In certain embodimentsthat may be combined with any of the preceding embodiments, the one ormore inflammatory cytokines are selected from the group consisting ofTNF-α, IL-10, IL-6, MCP-1, IFN-a4, IFN-b, IL-1β, IL-8, CRP, TGF-betamembers of the chemokine protein families, IL-20 family members, IL-33,LIF, IFN-gamma, OSM, CNTF, TGF-beta, GM-CSF, IL-11, IL-12, IL-17, IL-18,and CRP. In certain embodiments that may be combined with any of thepreceding embodiments, the one or more TREM2 activities, DAP12activities, or both comprise reduced expression of one or moreinflammatory receptors. In certain embodiments that may be combined withany of the preceding embodiments, the one or more inflammatory receptorscomprise CD86. In certain embodiments that may be combined with any ofthe preceding embodiments, the one or more TREM2 activities, DAP12activities, or both comprise increasing phagocytosis by macrophages,dendritic cells, monocytes, and/or microglia under conditions of reducedlevels of MCSF. In certain embodiments that may be combined with any ofthe preceding embodiments, the one or more TREM2 activities, DAP12activities, or both comprise decreasing phagocytosis by macrophages,dendritic cells, monocytes, and/or microglia in the presence of normallevels of MCSF. In certain embodiments that may be combined with any ofthe preceding embodiments, the one or more TREM2 activities, DAP12activities, or both comprise increasing activity of one or moreTREM2-dependent genes. In certain embodiments that may be combined withany of the preceding embodiments, the one or more TREM2-dependent genescomprise one or more nuclear factor of activated T-cells (NFAT)transcription factors. In certain embodiments that may be combined withany of the preceding embodiments, the antibody is of the IgG class theIgM class, or the IgA class. In certain embodiments that may be combinedwith any of the preceding embodiments, the antibody is of the IgG classand has an IgG1, IgG2, IgG3, or IgG4 isotype. In certain embodimentsthat may be combined with any of the preceding embodiments, the antibodyhas an IgG2 isotype. In certain embodiments that may be combined withany of the preceding embodiments, the antibody comprises a human IgG2constant region. In certain embodiments that may be combined with any ofthe preceding embodiments, the e human IgG2 constant region comprises anFc region. In certain embodiments that may be combined with any of thepreceding embodiments, the antibody induces the one or more TREM2activities, DAP12 activities, or both independently of binding to an Fcreceptor. In certain embodiments that may be combined with any of thepreceding embodiments, the antibody binds an inhibitory Fc receptor. Incertain embodiments that may be combined with any of the precedingembodiments, the inhibitory Fc receptor is inhibitory Fc-gamma receptorIIB (FcγIIB). In certain embodiments that may be combined with any ofthe preceding embodiments, the human IgG2 constant region comprises anFc region that comprises one or more modifications. In certainembodiments that may be combined with any of the preceding embodiments,the Fc region comprises one or more amino acid substitutions. In certainembodiments that may be combined with any of the preceding embodiments,the one or more amino acid substitutions in the Fc region are at aresidue position selected from the group consisting of V234A, G237A,H268Q, V309L, A330S, P331S, C232S, C233S, S267E, L328F, M252Y, S254T,T256E, and any combination thereof, wherein the numbering of theresidues is according to EU or Kabat numbering. In certain embodimentsthat may be combined with any of the preceding embodiments, the humanIgG2 constant region comprises a light chain constant region comprisinga C214S amino acid substitution, wherein the numbering of the residuesis according to EU or Kabat numbering. In certain embodiments that maybe combined with any of the preceding embodiments, the antibody has anIgG1 isotype. In certain embodiments that may be combined with any ofthe preceding embodiments, the antibody comprises a human IgG1 constantregion. In certain embodiments that may be combined with any of thepreceding embodiments, the human IgG1 constant region comprises an Fcregion. In certain embodiments that may be combined with any of thepreceding embodiments, the antibody binds an inhibitory Fc receptor. Incertain embodiments that may be combined with any of the precedingembodiments, the inhibitory Fc receptor is inhibitory Fc-gamma receptorIIB (FcγRIIB). In certain embodiments that may be combined with any ofthe preceding embodiments, the Fe region comprises one or moremodifications. In certain embodiments that may be combined with any ofthe preceding embodiments, the Fc region comprises one or more aminoacid substitutions. In certain embodiments that may be combined with anyof the preceding embodiments, the one or more amino acid substitutionsin the Fc region are at a residue position selected from the groupconsisting of N297A, D265A, L234A, L235A, G237A, C226S, C229S, E233P,L234V, L234F, L235E, P331S, S267E, L328F, A330L, M252Y, S254T, T256E,and any combination thereof, wherein the numbering of the residues isaccording to EU or Kabat numbering. In certain embodiments that may becombined with any of the preceding embodiments, the antibody comprisesan IgG2 isotype heavy chain constant domain 1(CH1) and hinge region. Incertain embodiments that may be combined with any of the precedingembodiments, the IgG2 isotype CH1 and hinge region comprise the aminoacid sequence of ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVT VPSSNFGTQT YTCNVDHKPSNTKVDKTVERKCCVECPPCP (SEQ ID NO: 397). In certain embodiments that maybe combined with any of the preceding embodiments, the antibody Fcregion comprises a S267E amino acid substitution, a L328F a L328F aminoacid substitution, or both, and/or a N297A or N297Q amino acidsubstitution, wherein the numbering of the residues on IgG1 is accordingto EU or Kabat numbering. In certain embodiments that may be combinedwith any of the preceding embodiments, the antibody comprises a mouseIgG1 constant region. In certain embodiments that may be combined withany of the preceding embodiments, the antibody has an IgG4 isotype. Incertain embodiments that may be combined with any of the precedingembodiments, the antibody comprises a human IgG4 constant region. Incertain embodiments that may be combined with any of the precedingembodiments, the human IgG4 constant region comprises an Fc region. Incertain embodiments that may be combined with any of the precedingembodiments, the antibody binds an inhibitory Fc receptor. In certainembodiments that may be combined with any of the preceding embodiments,the inhibitory Fc receptor is inhibitory Fc-gamma receptor IIB (FcγIIB).In certain embodiments that may be combined with any of the precedingembodiments, the Fc region comprises one or more modifications. Incertain embodiments that may be combined with any of the precedingembodiments, the Fc region comprises one or more amino acidsubstitutions. In certain embodiments that may be combined with any ofthe preceding embodiments, the one or more amino acid substitutions inthe Fc region are at a residue position selected from the groupconsisting of L235A, G237A, S228P, L236E, S267E, E318A, L328F, M252Y,S254T, T256E, and any combination thereof, wherein the numbering of theresidues is according to EU or Kabat numbering. In certain embodimentsthat may be combined with any of the preceding embodiments, the antibodyhas a hybrid IgG2/4 isotype. In certain embodiments that may be combinedwith any of the preceding embodiments, the antibody comprises an aminoacid sequence comprising amino acids 118 to 260 of human IgG2 and aminoacids 261 to 447 of human IgG4, wherein the numbering of the residues isaccording to EU or Kabat numbering. In certain embodiments that may becombined with any of the preceding embodiments, the antibody comprises amouse IgG4 constant region. In certain embodiments that may be combinedwith any of the preceding embodiments, the isolated antibody is anantibody fragment that binds to one or more human proteins selected fromthe group consisting of human TREM2, a naturally occurring variant ofhuman TREM2, human DAP12, and naturally occurring variant of humanDAP12, and wherein the antibody fragment is cross-linked to a secondantibody fragment that binds to one or more human proteins selected fromthe group consisting of human TREM2, a naturally occurring variant ofhuman TREM2, human DAP12, and naturally occurring variant of humanDAP12. In certain embodiments that may be combined with any of thepreceding embodiments, the fragment is an Fab, Fab′, Fab′-SH, F(ab′)2,Fv or scFv fragment.

Other aspects of the present disclosure relate to an isolated inertantibody that binds to a TREM2 protein. Other aspects of the presentdisclosure relate to an isolated antagonist antibody that binds to aTREM2 protein.

In certain embodiments that may be combined with any of the precedingembodiments, the TREM2 protein, the DAP12 protein, or both is amammalian protein or a human protein. In certain embodiments that may becombined with any of the preceding embodiments, the TREM2 protein, theDAP12 protein, or both is a wild-type protein. In certain embodimentsthat may be combined with any of the preceding embodiments, the TREM2protein, the DAP12 protein, or both is a naturally occurring variant. Incertain embodiments that may be combined with any of the precedingembodiments, the isolated antibody inhibits one or more TREM2activities, DAP12 activities, or both. In certain embodiments that maybe combined with any of the preceding embodiments, the one or more TREM2activities, DAP12 activities, or both comprise decreasing activity ofone or more TREM2-dependent genes. In certain embodiments that may becombined with any of the preceding embodiments, the one or moreTREM2-dependent genes comprise one or more nuclear factor of activatedT-cells (NFAT) transcription factors. In certain embodiments that may becombined with any of the preceding embodiments, the one or more TREM2activities, DAP12 activities, or both comprise decreasing the survivalof macrophages, microglial cells, M1 macrophages, M1 microglial cells,M2 macrophages, M2 microglial cells, osteoclasts, Langerhans cells ofskin, Kupffer cells, and/or dendritic cells. In certain embodiments thatmay be combined with any of the preceding embodiments, the isolatedantibody inhibits interaction between TREM2 and one or more TREM2ligands, inhibits TREM2 signal transduction, or both. In certainembodiments that may be combined with any of the preceding embodiments,the antibody is incapable of binding an Fc-gamma receptor (FcγR). Incertain embodiments that may be combined with any of the precedingembodiments, the antibody has an IgG1 isotype. In certain embodimentsthat may be combined with any of the preceding embodiments, the antibodycomprises a human IgG1 constant region. In certain embodiments that maybe combined with any of the preceding embodiments, the human IgG1constant region comprises an Fc region. In certain embodiments that maybe combined with any of the preceding embodiments, the Fc regioncomprises one or more modifications. In certain embodiments that may becombined with any of the preceding embodiments, the Fc region comprisesone or more amino acid substitutions. In certain embodiments that may becombined with any of the preceding embodiments, the one or more aminoacid substitutions in the Fc region are at a residue position selectedfrom the group consisting of N297A, N297Q, D265A, L234A, L235A, C226S,C229S, P238S, E233P, L234V, P238A, A327Q, A327G, P329A, K322A, L234F,L235E, P331S, T394D, A330L, M252Y, S254T, T256E, and any combinationthereof, wherein the numbering of the residues is according to EU orKabat numbering. In certain embodiments that may be combined with any ofthe preceding embodiments, the Fc region further comprises an amino aciddeletion at a position corresponding to glycine 236 according to EU orKabat numbering. In certain embodiments that may be combined with any ofthe preceding embodiments, the antibody comprises a mouse IgG1 constantregion. In certain embodiments that may be combined with any of thepreceding embodiments, the antibody has an IgG2 isotype. In certainembodiments that may be combined with any of the preceding embodiments,the antibody comprises a human IgG2 constant region. In certainembodiments that may be combined with any of the preceding embodiments,the e human IgG2 constant region comprises an Fc region. In certainembodiments that may be combined with any of the preceding embodiments,the Fc region comprises one or more modifications. In certainembodiments that may be combined with any of the preceding embodiments,the Fc region comprises one or more amino acid substitutions. In certainembodiments that may be combined with any of the preceding embodiments,the one or more amino acid substitutions in the Fc region are at aresidue position selected from the group consisting of V234A, G237A,H268E, V309L, N297A, N297Q, A330S, P331S, C232S, C233S, M252Y, S254T,T256E, and any combination thereof, wherein the numbering of theresidues is according to EU or Kabat numbering. In certain embodimentsthat may be combined with any of the preceding embodiments, the antibodyhas an IgG4 isotype. In certain embodiments that may be combined withany of the preceding embodiments, the antibody comprises a human IgG4constant region. In certain embodiments that may be combined with any ofthe preceding embodiments, the human IgG4 constant region comprises anFc region. In certain embodiments that may be combined with any of thepreceding embodiments, the Fc region comprises one or moremodifications. In certain embodiments that may be combined with any ofthe preceding embodiments, the Fc region comprises one or more aminoacid substitutions. In certain embodiments that may be combined with anyof the preceding embodiments, the one or more amino acid substitutionsin the Fc region are at a residue position selected from the groupconsisting of E233P, F234V, L235A, G237A, E318A, S228P, 1236E, S241P,1248E, T394D, M252Y, S254T, T256E, N297A, N297Q, and any combinationthereof, wherein the numbering of the residues is according to EU orKabat numbering. In certain embodiments that may be combined with any ofthe preceding embodiments, the isolated antibody is an antibody fragmentthat binds to one or more human proteins selected from the groupconsisting of human TREM2, a naturally occurring variant of human TREM2,human DAP12, and naturally occurring variant of human DAP12. In certainembodiments that may be combined with any of the preceding embodiments,the fragment is an Fab, Fab′, Fab′-SH, F(ab′)2, Fv or scFv fragment.

In certain embodiments that may be combined with any of the precedingembodiments, the Fc region further comprises one or more additionalamino acid substitutions at a position selected from the groupconsisting of A330L, L234F; L235E, P331S, and any combination thereof, wwherein the numbering of the residues is according to EU or Kabatnumbering. In certain embodiments that may be combined with any of thepreceding embodiments, the Fc region further comprises one or moreadditional amino acid substitutions at a position selected from thegroup consisting of M252Y, S254T, T256E, and any combination thereof,wherein the numbering of the residues is according to EU or Kabatnumbering. In certain embodiments that may be combined with any of thepreceding embodiments, the Fc region further comprises a S228P aminoacid substitution according to EU or Kabat numbering. In certainembodiments that may be combined with any of the preceding embodiments,the isolated antibody competes for binding of TREM2 with one or moreTREM2 ligands. In certain embodiments that may be combined with any ofthe preceding embodiments, the one or more TREM2 ligands are selectedfrom the group consisting of E. coli cells, apoptotic cells, nucleicacids, anionic lipids, zwitterionic lipids, negatively chargedphospholipids, phosphatidylserine, sulfatides, phosphatidylcholin,sphingomyelin, membrane phospholipids, lipidated proteins, proteolipids,lipidated peptides, and lipidated amyloid beta peptide. In certainembodiments that may be combined with any of the preceding embodiments,the isolated antibody is a human antibody, a humanized antibody, abispecific antibody, a multivalent antibody, a conjugated antibody, or achimeric antibody. In certain embodiments that may be combined with anyof the preceding embodiments, the isolated antibody is a bispecificantibody recognizing a first antigen and a second antigen. In certainembodiments that may be combined with any of the preceding embodiments,the first antigen is human TREM2 or a naturally occurring variantthereof, and the second antigen is a disease-causing protein selectedfrom the group consisting of amyloid beta or fragments thereof, Tau,IAPP, alpha-synuclein, TDP-43, FUS protein, prion protein, PrPSc,huntingtin, calcitonin, superoxide dismutase, ataxin, Lewy body, atrialnatriuretic factor, islet amyloid polypeptide, insulin, apolipoproteinAI, serum amyloid A, medin, prolactin, transthyretin, lysozyme, beta 2microglobulin, gelsolin, keratoepithelin, cystatin, immunoglobulin lightchain AL, S-IBM protein, Repeat-associated non-ATG (RAN) translationproducts, DiPeptide repeat (DPR) peptides, glycine-alanine (GA) repeatpeptides, glycine-proline (GP) repeat peptides, glycine-arginine (GR)repeat peptides, proline-alanine (PA) repeat peptides, andproline-arginine (PR) repeat peptides; a blood brain barrier targetingprotein selected from the group consisting of: trasnferin receptor,insulin receptor, insulin like growth factor receptor, LRP-1, and LRP1;or ligands and/or proteins expressed on immune cells. In certainembodiments that may be combined with any of the preceding embodiments,the isolated antibody is an antibody fragment that binds to one or morehuman proteins selected from the group consisting of human TREM2, anaturally occurring variant of human TREM2, human DAP12, and naturallyoccurring variant of human DAP12; and wherein the antibody is used incombination with one or more antibodies that specifically bind adisease-causing protein selected from the group consisting of: amyloidbeta or fragments thereof, Tau, IAPP, alpha-synuclein, TDP-43, FUSprotein, prion protein, PrPSc, huntingtin, calcitonin, superoxidedismutase, ataxin, Lewy body, atrial natriuretic factor, islet amyloidpolypeptide, insulin, apolipoprotein AI, serum amyloid A, medin,prolactin, transthyretin, lysozyme, beta 2 microglobulin, gelsolin,keratoepithelin, cystatin, immunoglobulin light chain AL, S-IBM protein,Repeat-associated non-ATG (RAN) translation products, DiPeptide repeat(DPR) peptides, glycine-alanine (GA) repeat peptides, glycine-proline(GP) repeat peptides, glycine-arginine (GR) repeat peptides,proline-alanine (PA) repeat peptides, and proline-arginine (PR) repeatpeptides, and any combination thereof. In certain embodiments that maybe combined with any of the preceding embodiments, the antibody is amonoclonal antibody.

In certain embodiments that may be combined with any of the precedingembodiments, the isolated antibody binds to a linear epitope on TREM2.In certain embodiments that may be combined with any of the precedingembodiments, the linear epitope on TREM2 is located within theextracellular domain of TREM2. In certain embodiments that may becombined with any of the preceding embodiments, the linear epitope onTREM2 is located within the extracellular immunoglobulin-likevariable-type (IgV) domain of TREM2. In certain embodiments that may becombined with any of the preceding embodiments, the isolated antibodybinds to a TREM2 protein, and wherein the isolated antibody binds to oneor more amino acids within amino acid residues selected from the groupconsisting of: i. amino acid residues 29-112 of SEQ ID NO: 1, or aminoacid residues on a TREM2 protein corresponding to amino acid residues29-112 of SEQ ID NO: 1; ii amino acid residues 29-41 of SEQ ID NO: 1, oramino acid residues on a TREM2 protein corresponding to amino acidresidues 29-41 of SEQ ID NO: 1; iii. amino acid residues 40-44 of SEQ IDNO: 1, or amino acid residues on a TREM2 protein corresponding to aminoacid residues 40-44 of SEQ ID NO: 1; iv. amino acid residues 47-69 ofSEQ ID NO: 1, or amino acid residues on a TREM2 protein corresponding toamino acid residues 47-69 of SEQ ID NO: 1; v. amino acid residues 67-76of SEQ ID NO: 1, or amino acid residues on a TREM2 protein correspondingto amino acid residues 67-76 of SEQ ID NO: 1; vi. amino acid residues76-86 of SEQ ID NO: 1, or amino acid residues on a TREM2 proteincorresponding to amino acid residues 76-86 of SEQ ID NO: 1; vii. aminoacid residues 91-100 of SEQ ID NO: 1, or amino acid residues on a TREM2protein corresponding to amino acid residues 91-100 of SEQ ID NO: 1;viii. amino acid residues 99-115 of SEQ ID NO: 1, or amino acid residueson a TREM2 protein corresponding to amino acid residues 99-115 of SEQ IDNO: 1; ix. amino acid residues 104-112 of SEQ ID NO: 1, or amino acidresidues on a TREM2 protein corresponding to amino acid residues 104-112of SEQ ID NO: 1; and x. amino acid residues 114-118 of SEQ ID NO: 1, oramino acid residues on a TREM2 protein corresponding to amino acidresidues 114-118 of SEQ ID NO: 1. In certain embodiments that may becombined with any of the preceding embodiments, the isolated antibodybinds to one or more amino acids within amino acid residues 43-50 of SEQID NO: 1, or amino acid residues on a TREM2 protein corresponding toamino acid residues 43-50 of SEQ ID NO: 1. In certain embodiments thatmay be combined with any of the preceding embodiments, the isolatedantibody binds to one or more amino acids within amino acid residues49-57 of SEQ ID NO: 1, or amino acid residues on a TREM2 proteincorresponding to amino acid residues 49-57 of SEQ ID NO: 1. In certainembodiments that may be combined with any of the preceding embodiments,the isolated antibody binds to an epitope comprising one or more aminoacids within amino acid residues 43-50 of SEQ ID NO: 1. In certainembodiments that may be combined with any of the preceding embodiments,the isolated antibody binds to an epitope comprising one or more aminoacids within amino acid residues 43-50 of SEQ ID NO: 1. In certainembodiments that may be combined with any of the preceding embodiments,the isolated antibody binds to an epitope comprising one or more aminoacid residues selected from the group consisting of: i. amino acidresidue Arg47 or Asp87 of SEQ ID NO: 1; ii. amino acid residues 40-44 ofSEQ ID NO: 1; iii. amino acid residues 67-76 of SEQ ID NO: 1; and iv.amino acid residues 114-118 of SEQ ID NO: 1. In certain embodiments thatmay be combined with any of the preceding embodiments, the isolatedantibody binds to one or more amino acids within amino acid residues22-40 of SEQ ID NO: 2, or amino acid residues on a DAP12 proteincorresponding to amino acid residues 22-40 of SEQ ID NO: 2. In certainembodiments that may be combined with any of the preceding embodiments,the isolated antibody is a bispecific antibody that binds to one or moreamino acids selected from the group consisting of: i. one or more aminoacid residues of SEQ ID NO: 1, or amino acid residues on a TREM2 proteincorresponding to amino acid residues of SEQ ID NO: 1; and ii. one ormore amino acid residues of SEQ ID NO: 2, or amino acid residues on aDAP12 protein corresponding to amino acid residues of SEQ ID NO: 2.

In certain embodiments that may be combined with any of the precedingembodiments, the isolated antibody comprises a heavy chain variabledomain and a light chain variable domain, wherein the heavy chainvariable domain comprises the HVR-H1, HVR-H2, and/or HVR-H3 of themonoclonal antibody Ab52; and/or wherein the light chain variable domaincomprises the HVR-L1, HVR-L2, and/or HVR-L3 of the monoclonal antibodyAb52. In certain embodiments that may be combined with any of thepreceding embodiments, the HVR-H1 comprises the amino acid sequence ofSEQ ID NO:398. In certain embodiments that may be combined with any ofthe preceding embodiments, the HVR-H2 comprises the amino acid sequenceof SEQ ID NO:399. In certain embodiments that may be combined with anyof the preceding embodiments, the HVR-H3 comprises the amino acidsequence of SEQ ID NO:400. In certain embodiments that may be combinedwith any of the preceding embodiments, the HVR-L1 comprises the aminoacid sequence of SEQ ID NO:401. In certain embodiments that may becombined with any of the preceding embodiments, the HVR-L2 comprises theamino acid sequence of SEQ ID NO:402. In certain embodiments that may becombined with any of the preceding embodiments, the HVR-L3 comprises theamino acid sequence of SEQ ID NO:403. In certain embodiments that may becombined with any of the preceding embodiments, the isolated antibodycomprises a heavy chain variable domain and a light chain variabledomain, wherein the heavy chain variable domain comprises: (a) an HVR-H1comprising the amino acid sequence of SEQ ID NO:398, or an amino acidsequence with at least about 95% homology to the amino acid sequence ofSEQ ID NO:398; (b) an HVR-H2 comprising the amino acid sequence of SEQID NO:399, or an amino acid sequence with at least about 95% homology tothe amino acid sequence of SEQ ID NO:399; and; and/or (c) an HVR-H3comprising the amino acid sequence of SEQ ID NO:400, or an amino acidsequence with at least about 95% homology to the amino acid sequence ofSEQ ID NO:400; and/or wherein the light chain variable domain comprises:(a) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:401, or anamino acid sequence with at least about 95% homology to the amino acidsequence of SEQ ID NO:401; (b) an HVR-L2 comprising the amino acidsequence of SEQ ID NO:402, or an amino acid sequence with at least about95% homology to the amino acid sequence of SEQ ID NO:402; and/or (c) anHVR-L3 comprising the amino acid sequence of SEQ ID NO:403, or an aminoacid sequence with at least about 95% homology to the amino acidsequence of SEQ ID NO:403. In certain embodiments that may be combinedwith any of the preceding embodiments, the isolated antibody comprises aheavy chain variable domain and a light chain variable domain, whereinthe heavy chain variable domain comprises the HVR-H1, HVR-H2, and/orHVR-H3 of the monoclonal antibody Ab21; and/or wherein the light chainvariable domain comprises the HVR-L1, HVR-L2, and/or HVR-L3 of themonoclonal antibody Ab21. In certain embodiments that may be combinedwith any of the preceding embodiments, the HVR-H1 comprises the aminoacid sequence of SEQ ID NO:404. In certain embodiments that may becombined with any of the preceding embodiments, the HVR-H2 comprises theamino acid sequence of SEQ ID NO:405. In certain embodiments that may becombined with any of the preceding embodiments, the HVR-H3 comprises theamino acid sequence of SEQ ID NO:406. In certain embodiments that may becombined with any of the preceding embodiments, the HVR-L1 comprises theamino acid sequence of SEQ ID NO:407. In certain embodiments that may becombined with any of the preceding embodiments, the HVR-L2 comprises theamino acid sequence of SEQ ID NO:408. In certain embodiments that may becombined with any of the preceding embodiments, the HVR-L3 comprises theamino acid sequence of SEQ ID NO:409. In certain embodiments that may becombined with any of the preceding embodiments, the isolated antibodycomprises a heavy chain variable domain and a light chain variabledomain, wherein the heavy chain variable domain comprises: (a) n HVR-H1comprising the amino acid sequence of SEQ ID NO:404, or an amino acidsequence with at least about 95% homology to the amino acid sequence ofSEQ ID NO:404; (b) an HVR-H2 comprising the amino acid sequence of SEQID NO:405, or an amino acid sequence with at least about 95% homology tothe amino acid sequence of SEQ ID NO:405; and/or (c) an HVR-H3comprising the amino acid sequence of SEQ ID NO:406, or an amino acidsequence with at least about 95% homology to the amino acid sequence ofSEQ ID NO:406, and/or wherein the light chain variable domain comprises:(a) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:407, or anamino acid sequence with at least about 95% homology to the amino acidsequence of SEQ ID NO:407; (b) an HVR-L2 comprising the amino acidsequence of SEQ ID NO:408, or an amino acid sequence with at least about95% homology to the amino acid sequence of SEQ ID NO:408; and/or (c) anHVR-L3 comprising the amino acid sequence of SEQ ID NO:409, or an aminoacid sequence with at least about 95% homology to the amino acidsequence of SEQ ID NO:409.

Other aspects of the present disclosure relate to an isolated anti-humanTREM2 antibody, wherein the isolated antibody comprises a heavy chainvariable domain and a light chain variable domain, wherein the heavychain variable domain comprises the HVR-H1, HVR-H2, and/or HVR-H3 of themonoclonal antibody Ab52; and/or wherein the light chain variable domaincomprises the HVR-L1, HVR-L2, and/or HVR-L3 of the monoclonal antibodyAb52. In certain embodiments that may be combined with any of thepreceding embodiments, the HVR-H1 comprises the amino acid sequence ofSEQ ID NO:398. In certain embodiments that may be combined with any ofthe preceding embodiments, the HVR-H2 comprises the amino acid sequenceof SEQ ID NO:399. In certain embodiments that may be combined with anyof the preceding embodiments, the HVR-H3 comprises the amino acidsequence of SEQ ID NO:400. In certain embodiments that may be combinedwith any of the preceding embodiments, the HVR-L1 comprises the aminoacid sequence of SEQ ID NO:401. In certain embodiments that may becombined with any of the preceding embodiments, the HVR-L2 comprises theamino acid sequence of SEQ ID NO:402. In certain embodiments that may becombined with any of the preceding embodiments, the HVR-L3 comprises theamino acid sequence of SEQ ID NO:403. In certain embodiments that may becombined with any of the preceding embodiments, the isolated antibodycomprises a heavy chain variable domain and a light chain variabledomain, wherein the heavy chain variable domain comprises an HVR-H1comprising the amino acid sequence of SEQ ID NO:398, an HVR-H2comprising the amino acid sequence of SEQ ID NO:399, and an HVR-H3comprising the amino acid sequence of SEQ ID NO:400, and/or wherein thelight chain variable domain comprises an HVR-L1 comprising the aminoacid sequence of SEQ ID NO:401, an HVR-L2 comprising the amino acidsequence of SEQ ID NO:402, and an HVR-L3 comprising the amino acidsequence of SEQ ID NO:403.

Other aspects of the present disclosure relate to an isolated anti-humanTREM2 antibody, wherein the isolated antibody comprises a heavy chainvariable domain and a light chain variable domain, wherein the heavychain variable domain comprises: (a) an HVR-H1 comprising the amino acidsequence of SEQ ID NO:398, or an amino acid sequence with at least about95% homology to the amino acid sequence of SEQ ID NO:398; (b) an HVR-H2comprising the amino acid sequence of SEQ ID NO:399, or an amino acidsequence with at least about 95% homology to the amino acid sequence ofSEQ ID NO:399; and; and/or (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO:400, or an amino acid sequence with at least about95% homology to the amino acid sequence of SEQ ID NO:400; and/or whereinthe light chain variable domain comprises: (a) an HVR-L1 comprising theamino acid sequence of SEQ ID NO:401, or an amino acid sequence with atleast about 95% homology to the amino acid sequence of SEQ ID NO:401;(b) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:402, or anamino acid sequence with at least about 95% homology to the amino acidsequence of SEQ ID NO:402; and/or (c) an HVR-L3 comprising the aminoacid sequence of SEQ ID NO:403, or an amino acid sequence with at leastabout 95% homology to the amino acid sequence of SEQ ID NO:403.

Other aspects of the present disclosure relate to an isolated anti-humanTREM2 antibody, wherein the isolated antibody comprises a heavy chainvariable domain and a light chain variable domain, wherein the heavychain variable domain comprises the HVR-H1, HVR-H2, and/or HVR-H3 of themonoclonal antibody Ab21; and/or wherein the light chain variable domaincomprises the HVR-L1, HVR-L2, and/or HVR-L3 of the monoclonal antibodyAb21. In certain embodiments that may be combined with any of thepreceding embodiments, the HVR-H1 comprises the amino acid sequence ofSEQ ID NO:404. In certain embodiments that may be combined with any ofthe preceding embodiments, the HVR-H2 comprises the amino acid sequenceof SEQ ID NO:405. In certain embodiments that may be combined with anyof the preceding embodiments, the HVR-H3 comprises the amino acidsequence of SEQ ID NO:406. In certain embodiments that may be combinedwith any of the preceding embodiments, the HVR-L1 comprises the aminoacid sequence of SEQ ID NO:407. In certain embodiments that may becombined with any of the preceding embodiments, the HVR-L2 comprises theamino acid sequence of SEQ ID NO:408. In certain embodiments that may becombined with any of the preceding embodiments, the HVR-L3 comprises theamino acid sequence of SEQ ID NO:409. In certain embodiments that may becombined with any of the preceding embodiments, the isolated antibodycomprises a heavy chain variable domain and a light chain variabledomain, wherein the heavy chain variable domain comprises an HVR-H1comprising the amino acid sequence of SEQ ID NO:404, an HVR-H2comprising the amino acid sequence of SEQ ID NO:405, and an HVR-H3comprising the amino acid sequence of SEQ ID NO:406, and/or wherein thelight chain variable domain comprises an HVR-L1 comprising the aminoacid sequence of SEQ ID NO:407, an HVR-L2 comprising the amino acidsequence of SEQ ID NO:408, and an HVR-L3 comprising the amino acidsequence of SEQ ID NO:409.

Other aspects of the present disclosure relate to an isolated anti-humanTREM2 antibody, wherein the isolated antibody comprises a heavy chainvariable domain and a light chain variable domain, wherein the heavychain variable domain comprises: (a) n HVR-H1 comprising the amino acidsequence of SEQ ID NO:404, or an amino acid sequence with at least about95% homology to the amino acid sequence of SEQ ID NO:404; (b) an HVR-H2comprising the amino acid sequence of SEQ ID NO:405, or an amino acidsequence with at least about 95% homology to the amino acid sequence ofSEQ ID NO:405; and/or (c) an HVR-H3 comprising the amino acid sequenceof SEQ ID NO:406, or an amino acid sequence with at least about 95%homology to the amino acid sequence of SEQ ID NO:406, and/or wherein thelight chain variable domain comprises: (a) an HVR-L1 comprising theamino acid sequence of SEQ ID NO:407, or an amino acid sequence with atleast about 95% homology to the amino acid sequence of SEQ ID NO:407;(b) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:408, or anamino acid sequence with at least about 95% homology to the amino acidsequence of SEQ ID NO:408; and/or (c) an HVR-L3 comprising the aminoacid sequence of SEQ ID NO:409, or an amino acid sequence with at leastabout 95% homology to the amino acid sequence of SEQ ID NO:409.

Other aspects of the present disclosure relate to an isolated anti-humanTREM2 antibody which binds essentially the same TREM2 epitope as theantibody Ab52. Other aspects of the present disclosure relate to anisolated anti-human TREM2 antibody which binds essentially the sameTREM2 epitope as the antibody Ab21.

In certain embodiments that may be combined with any of the precedingembodiments, the antibody is an agonist antibody, and wherein theantibody induces one or more TREM2 activities, DAP12 activities, orboth. In certain embodiments that may be combined with any of thepreceding embodiments, the isolated antibody induces or retains TREM2clustering, DAP12 clustering, or both on a cell surface. In certainembodiments that may be combined with any of the preceding embodiments,the one or more TREM2 activities, DAP12 activities, or both are selectedfrom the group consisting of TREM2 binding to DAP12; DAP12 binding toTREM2; TREM2 phosphorylation, DAP12 phosphorylation; PI3K activation;increased expression of one or more anti-inflammatory mediators (e.g.,cytokines) selected from the group consisting of IL-12p70, IL-6, andIL-10; reduced expression of one or more pro-inflammatory mediatorsselected from the group consisting of IFN-a4, IFN-b, IL-6, IL-12 p70,IL-1β, TNF, TNF-α, IL-10, IL-8, CRP, TGF-beta members of the chemokineprotein families, IL-20 family members, IL-33, LIF, IFN-gamma, OSM,CNTF, TGF-beta, GM-CSF, IL-11, IL-12, IL-17, IL-18, and CRP; reducedexpression of TNF-α, IL-6, or both; extracellular signal-regulatedkinase (ERK) phosphorylation; increased expression of C-C chemokinereceptor 7 (CCR7); induction of microglial cell chemotaxis toward CCL19and CCL21 expressing cells; an increase, normalization, or both of theability of bone marrow-derived dendritic cells to induceantigen-specific T-cell proliferation; induction of osteoclastproduction, increased rate of osteoclastogenesis, or both; increasingthe survival and/or function of one or more of dendritic cells,macrophages, microglial cells, M1 macrophages and/or microglial cells,activated M1 macrophages and/or microglial cells, M2 macrophages and/ormicroglial cells, monocytes, osteoclasts, Langerhans cells of skin, andKupffer cells; induction of one or more types of clearance selected fromthe group consisting of apoptotic neuron clearance, nerve tissue debrisclearance, non-nerve tissue debris clearance, bacteria or other foreignbody clearance, disease-causing protein clearance, disease-causingpeptide clearance, and disease-causing nucleic acid clearance; inductionof phagocytosis of one or more of apoptotic neurons, nerve tissuedebris, non-nerve tissue debris, bacteria, other foreign bodies,disease-causing proteins, disease-causing peptides, or disease-causingnucleic acids; normalization of disrupted TREM2/DAP12-dependent geneexpression; recruitment of Syk, ZAP70, or both to the TREM2/DAP12complex; Syk phosphorylation; increased expression of CD83 and/or CD86on dendritic cells, macrophages, monocytes, and/or microglia; reducedsecretion of one or more inflammatory cytokines selected from the groupconsisting of TNF-α, IL-10, IL-6, MCP-1, IFN-a4, IFN-b, IL-1β, IL-8,CRP, TGF-beta members of the chemokine protein families, IL-20 familymembers, IL-33, LIF, IFN-gamma, OSM, CNTF, TGF-beta, GM-CSF, IL-11,IL-12, IL-17, IL-18, and CRP; reduced expression of one or moreinflammatory receptors; increasing phagocytosis by macrophages,dendritic cells, monocytes, and/or microglia under conditions of reducedlevels of MCSF; decreasing phagocytosis by macrophages, dendritic cells,monocytes, and/or microglia in the presence of normal levels of MCSF;increasing activity of one or more TREM2-dependent genes; and anycombination thereof. In certain embodiments that may be combined withany of the preceding embodiments, the antibody is of the IgG class theIgM class, or the IgA class. In certain embodiments that may be combinedwith any of the preceding embodiments, the antibody is of the IgG classand has an IgG1, IgG2, IgG3, or IgG4 isotype. In certain embodimentsthat may be combined with any of the preceding embodiments, the antibodyhas an IgG2 isotype. In certain embodiments that may be combined withany of the preceding embodiments, the antibody comprises a human IgG2constant region. In certain embodiments that may be combined with any ofthe preceding embodiments, the human IgG2 constant region comprises anFc region. In certain embodiments that may be combined with any of thepreceding embodiments, the antibody induces the one or more TREM2activities, DAP12 activities, or both independently of binding to an Fcreceptor. In certain embodiments that may be combined with any of thepreceding embodiments, the antibody binds an inhibitory Fc receptor. Incertain embodiments that may be combined with any of the precedingembodiments, the inhibitory Fc receptor is inhibitory Fc-gamma receptorIIB (FcγIIB). In certain embodiments that may be combined with any ofthe preceding embodiments, the Fc region comprises one or moremodifications. In certain embodiments that may be combined with any ofthe preceding embodiments, the Fc region comprises one or more aminoacid substitutions. In certain embodiments that may be combined with anyof the preceding embodiments, the one or more amino acid substitutionsin the Fc region are at a residue position selected from the groupconsisting of V234A, G237A, H268Q, V309L, A330S, P331S, C232S, C233S,S267E, L328F, M252Y, S254T, T256E, and any combination thereof, whereinthe numbering of the residues is according to EU or Kabat numbering. Incertain embodiments that may be combined with any of the precedingembodiments, the human IgG2 constant region comprises a light chainconstant region comprising a C214S amino acid substitution, wherein thenumbering of the residues is according to EU or Kabat numbering. Incertain embodiments that may be combined with any of the precedingembodiments, the antibody has an IgG1 isotype. In certain embodimentsthat may be combined with any of the preceding embodiments, the antibodycomprises a human IgG1 constant region. In certain embodiments that maybe combined with any of the preceding embodiments, the human IgG1constant region comprises an Fc region. In certain embodiments that maybe combined with any of the preceding embodiments, the antibody binds aninhibitory Fc receptor. In certain embodiments that may be combined withany of the preceding embodiments, the inhibitory Fc receptor isinhibitory Fc-gamma receptor IIB (FcγRIIB). In certain embodiments thatmay be combined with any of the preceding embodiments, the Fc regioncomprises one or more modifications. In certain embodiments that may becombined with any of the preceding embodiments, the Fc region comprisesone or more amino acid substitutions. In certain embodiments that may becombined with any of the preceding embodiments, the one or more aminoacid substitutions in the Fc region are at a residue position selectedfrom the group consisting of N297A, D265A, L234A, L235A, G237A, C226S,C229S, E233P, L234V, L234F, L235E, P331S, S267E, L328F, A330L, M252Y,S254T, T256E, and any combination thereof, wherein the numbering of theresidues is according to EU or Kabat numbering. In certain embodimentsthat may be combined with any of the preceding embodiments, the antibodycomprises an IgG2 isotype heavy chain constant domain 1(CH1) and hingeregion. In certain embodiments that may be combined with any of thepreceding embodiments, the IgG2 isotype CH1 and hinge region comprisethe amino acid sequence of ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVT VPSSNFGTQT YTCNVDHKPSNTKVDKTVERKCCVECPPCP (SEQ ID NO: 397). In certain embodiments that maybe combined with any of the preceding embodiments, the antibody Fcregion comprises a S267E amino acid substitution, a L328F amino acidsubstitution, or both, and/or a N297A or N297Q amino acid substitution,wherein the numbering of the residues is according to EU or Kabatnumbering. In certain embodiments that may be combined with any of thepreceding embodiments, the antibody comprises a mouse IgG1 constantregion. In certain embodiments that may be combined with any of thepreceding embodiments, the antibody has an IgG4 isotype. In certainembodiments that may be combined with any of the preceding embodiments,the antibody comprises a human IgG4 constant region. In certainembodiments that may be combined with any of the preceding embodiments,the human IgG4 constant region comprises an Fc region. In certainembodiments that may be combined with any of the preceding embodiments,the antibody binds an inhibitory Fc receptor. In certain embodimentsthat may be combined with any of the preceding embodiments, theinhibitory Fc receptor is inhibitory Fc-gamma receptor IIB (FcγIIB). Incertain embodiments that may be combined with any of the precedingembodiments, the Fc region comprises one or more modifications. Incertain embodiments that may be combined with any of the precedingembodiments, the Fc region comprises one or more amino acidsubstitutions. In certain embodiments that may be combined with any ofthe preceding embodiments, the one or more amino acid substitutions inthe Fe region are at a residue position selected from the groupconsisting of L235A, G237A, S228P, L236E, S267E, E318A, L328F, M252Y,S254T, T256E, and any combination thereof, wherein the numbering of theresidues is according to EU or Kabat numbering. In certain embodimentsthat may be combined with any of the preceding embodiments, the antibodyhas a hybrid IgG2/4 isotype. In certain embodiments that may be combinedwith any of the preceding embodiments, the antibody comprises an aminoacid sequence comprising amino acids 118 to 260 of human IgG2 and aminoacids 261 to 447 of human IgG4, wherein the numbering of the residues isaccording to EU or Kabat numbering. In certain embodiments that may becombined with any of the preceding embodiments, the antibody comprises amouse IgG4 constant region. In certain embodiments that may be combinedwith any of the preceding embodiments, the isolated antibody is anantibody fragment that binds to one or more human proteins selected fromthe group consisting of human TREM2, a naturally occurring variant ofhuman TREM2, human DAP12, and naturally occurring variant of humanDAP12, and wherein the antibody fragment is cross-linked to a secondantibody fragment that binds to one or more human proteins selected fromthe group consisting of human TREM2, a naturally occurring variant ofhuman TREM2, human DAP12, and naturally occurring variant of humanDAP12. In certain embodiments that may be combined with any of thepreceding embodiments, the fragment is an Fab, Fab′, Fab′-SH, F(ab′)2,Fv or scFv fragment. In certain embodiments that may be combined withany of the preceding embodiments, the isolated antibody is an inertantibody. In certain embodiments that may be combined with any of thepreceding embodiments, the isolated antibody is an antagonist antibody.In certain embodiments that may be combined with any of the precedingembodiments, the isolated antibody inhibits one or more TREM2activities. In certain embodiments that may be combined with any of thepreceding embodiments, the one or more TREM2 activities are selectedfrom the group consisting of decreasing activity of one or moreTREM2-dependent genes; decreasing activity of one or more nuclear factorof activated T-cells (NFAT) transcription factors; decreasing thesurvival of macrophages, microglial cells, monocytes, osteoclasts,Langerhans cells of skin, Kupffer cells, and/or dendritic cells; and anycombination thereof. In certain embodiments that may be combined withany of the preceding embodiments, the isolated antibody inhibitsinteraction between TREM2 and one or more TREM2 ligands, inhibits TREM2signal transduction, or both. In certain embodiments that may becombined with any of the preceding embodiments, the antibody isincapable of binding an Fc-gamma receptor (FcγR). In certain embodimentsthat may be combined with any of the preceding embodiments, the antibodyhas an IgG1 isotype. In certain embodiments that may be combined withany of the preceding embodiments, the antibody comprises a human IgG1constant region. In certain embodiments that may be combined with any ofthe preceding embodiments, the human IgG1 constant region comprises anFc region. In certain embodiments that may be combined with any of thepreceding embodiments, the Fc region comprises one or moremodifications. In certain embodiments that may be combined with any ofthe preceding embodiments, the Fc region comprises one or more aminoacid substitutions. In certain embodiments that may be combined with anyof the preceding embodiments, the one or more amino acid substitutionsin the Fc region are at a residue position selected from the groupconsisting of N297A, N297Q, D265A, L234A, L235A, C226S, C229S, P238S,E233P, L234V, P238A, A327Q, A327G, P329A, K322A, L234F, L235E, P331S,T394D, A330L, M252Y, S254T, T256E, and any combination thereof, whereinthe numbering of the residues is according to EU or Kabat numbering. Incertain embodiments that may be combined with any of the precedingembodiments, the Fc region further comprises an amino acid deletion at aposition corresponding to glycine 236 according to EU or Kabatnumbering. In certain embodiments that may be combined with any of thepreceding embodiments, the antibody comprises a mouse IgG1 constantregion. In certain embodiments that may be combined with any of thepreceding embodiments, the antibody has an IgG2 isotype. In certainembodiments that may be combined with any of the preceding embodiments,the antibody comprises a human IgG2 constant region. In certainembodiments that may be combined with any of the preceding embodiments,the human IgG2 constant region comprises an Fc region. In certainembodiments that may be combined with any of the preceding embodiments,the Fc region comprises one or more modifications. In certainembodiments that may be combined with any of the preceding embodiments,the Fc region comprises one or more amino acid substitutions. In certainembodiments that may be combined with any of the preceding embodiments,the one or more amino acid substitutions in the Fc region are at aresidue position selected from the group consisting of V234A, G237A,H268E, V309L, N297A, N297Q, A330S, P331S, C232S, C233S, M252Y, S254T,T256E, and any combination thereof, wherein the numbering of theresidues is according to EU or Kabat numbering. In certain embodimentsthat may be combined with any of the preceding embodiments, the antibodyhas an IgG4 isotype. In certain embodiments that may be combined withany of the preceding embodiments, the antibody comprises a human IgG4constant region. In certain embodiments that may be combined with any ofthe preceding embodiments, the human IgG4 constant region comprises anFc region. In certain embodiments that may be combined with any of thepreceding embodiments, the Fc region comprises one or moremodifications. In certain embodiments that may be combined with any ofthe preceding embodiments, the Fc region comprises one or more aminoacid substitutions. In certain embodiments that may be combined with anyof the preceding embodiments, the one or more amino acid substitutionsin the Fc region are at a residue position selected from the groupconsisting of E233P, F234V, L235A, G237A, E318A, S228P, L236E, S241P,L248E, T394D, M252Y, S254T, T256E, N297A, N297Q, and any combinationthereof, wherein the numbering of the residues is according to EU orKabat numbering. In certain embodiments that may be combined with any ofthe preceding embodiments, the isolated antibody is an antibody fragmentthat binds to one or more human proteins selected from the groupconsisting of human TREM2, a naturally occurring variant of human TREM2,human DAP12, and naturally occurring variant of human DAP12. In certainembodiments that may be combined with any of the preceding embodiments,the fragment is an Fab, Fab′, Fab′-SH, F(ab′)2, Fv or scFv fragment. Incertain embodiments that may be combined with any of the precedingembodiments, the Fc region further comprises one or more additionalamino acid substitutions at a position selected from the groupconsisting of A330L, L234F; L235E, P331S, and any combination thereof,wherein the numbering of the residues is according to EU or Kabatnumbering. In certain embodiments that may be combined with any of thepreceding embodiments, the Fc region further comprises one or moreadditional amino acid substitutions at a position selected from thegroup consisting of M252Y, S254T, T256E, and any combination thereof,wherein the numbering of the residues is according to EU or Kabatnumbering. In certain embodiments that may be combined with any of thepreceding embodiments, the Fc region further comprises a S228P aminoacid substitution according to EU or Kabat numbering. In certainembodiments that may be combined with any of the preceding embodiments,the antibody is a human antibody, a humanized antibody, a bispecificantibody, a multivalent antibody, or a chimeric antibody. In certainembodiments that may be combined with any of the preceding embodiments,the antibody is a bispecific antibody recognizing a first antigen and asecond antigen. In certain embodiments that may be combined with any ofthe preceding embodiments, the antibody is a monoclonal antibody.

Other aspects of the present disclosure relate to an isolated antibodythat binds to a TREM2 protein, wherein the isolated antibody promotessurvival of one or more innate immune cells. Other aspects of thepresent disclosure relate to an isolated antibody that binds to a TREM2protein, wherein the isolated antibody increases expression of IL-6.Other aspects of the present disclosure relate to an isolated antibodythat binds to a TREM2 protein, wherein the isolated antibody promotessurvival of one or more innate immune cells or increases expression ofIL-6. Other aspects of the present disclosure relate to an isolatedantibody that binds to a TREM2 protein, wherein the isolated antibodypromotes survival of one or more innate immune cells and increasesexpression of IL-6. In certain embodiments, the one or more innateimmune cells are selected from the group consisting of macrophages,microglial cells, M1 microglial cells, activated M1 microglial cells, M2microglial cells, dendritic cells, M1 macrophages, activated M1macrophages, M2 macrophages, monocytes, osteoclasts, Langerhans cells ofskin, Kupffer cells, and any combination thereof. In certainembodiments, the one or more innate immune cells are macrophages. Incertain embodiments, the one or more innate immune cells are microglialcells. In certain embodiments, the one or more innate immune cells areM1 microglial cells. In certain embodiments, the one or more innateimmune cells are activated M1 microglial cells. In certain embodiments,the one or more innate immune cells are M2 microglial cells. In certainembodiments, the one or more innate immune cells are dendritic cells(DCs). In certain embodiments, the one or more innate immune cells areM1 macrophages. In certain embodiments, the one or more innate immunecells are activated M1 macrophages. In certain embodiments, the one ormore innate immune cells are M2 macrophages. In certain embodiments, theone or more innate immune cells are monocytes. In certain embodiments,the one or more innate immune cells are osteoclasts. In certainembodiments, the one or more innate immune cells are Langerhans cells ofskin. In certain embodiments, the one or more innate immune cells areKupffer cells.

Other aspects of the present disclosure relate to an isolated antibodythat binds to a TREM2 protein, wherein the isolated antibody binds toone or more amino acids within amino acid residues selected from thegroup consisting of: i. amino acid residues 29-112 of SEQ ID NO: 1, oramino acid residues on a TREM2 protein corresponding to amino acidresidues 29-112 of SEQ ID NO: 1; ii. amino acid residues 29-41 of SEQ IDNO: 1, or amino acid residues on a TREM2 protein corresponding to aminoacid residues 29-41 of SEQ ID NO: 1; iii. amino acid residues 40-44 ofSEQ ID NO: 1, or amino acid residues on a TREM2 protein corresponding toamino acid residues 40-44 of SEQ ID NO: 1; iv. amino acid residues 43-50of SEQ ID NO: 1, or amino acid residues on a TREM2 protein correspondingto amino acid residues 43-50 of SEQ ID NO: 1; v. amino acid residues49-57 of SEQ ID NO: 1, or amino acid residues on a TREM2 proteincorresponding to amino acid residues 49-57 of SEQ ID NO: 1; vi. aminoacid residues 47-69 of SEQ ID NO: 1, or amino acid residues on a TREM2protein corresponding to amino acid residues 47-69 of SEQ ID NO: 1; vii.amino acid residues 67-76 of SEQ ID NO: 1, or amino acid residues on aTREM2 protein corresponding to amino acid residues 67-76 of SEQ ID NO:1; viii. amino acid residues 76-86 of SEQ ID NO: 1, or amino acidresidues on a TREM2 protein corresponding to amino acid residues 76-86of SEQ ID NO: 1; ix. amino acid residues 91-100 of SEQ ID NO: 1, oramino acid residues on a TREM2 protein corresponding to amino acidresidues 91-100 of SEQ ID NO: 1; x. amino acid residues 99-115 of SEQ IDNO: 1, or amino acid residues on a TREM2 protein corresponding to aminoacid residues 99-115 of SEQ ID NO: 1; xi. amino acid residues 104-112 ofSEQ ID NO: 1, or amino acid residues on a TREM2 protein corresponding toamino acid residues 104-112 of SEQ ID NO: 1; xii. amino acid residues114-118 of SEQ ID NO: 1, or amino acid residues on a TREM2 proteincorresponding to amino acid residues 114-118 of SEQ ID NO: 1; xiii.amino acid residues 130-171 of SEQ ID NO: 1, or amino acid residues on aTREM2 protein corresponding to amino acid residues 130-171 of SEQ ID NO:1; xiv. amino acid residues 139-146 of SEQ ID NO: 1, or amino acidresidues on a TREM2 protein corresponding to amino acid residues 139-146of SEQ ID NO: 1; xv. amino acid residues 140-153 of SEQ ID NO: 1, oramino acid residues on a TREM2 protein corresponding to amino acidresidues 140-153 of SEQ ID NO: 1; xvi. amino acid residues 130-144 ofSEQ ID NO: 1, or amino acid residues on a TREM2 protein corresponding toamino acid residues 130-144 of SEQ ID NO: 1; and xvii. amino acidresidues 158-171 of SEQ ID NO: 1, or amino acid residues on a TREM2protein corresponding to amino acid residues 158-171 of SEQ ID NO: 1. Incertain embodiments, the isolated antibody binds to one or more aminoacids within amino acid residues 43-50 of SEQ ID NO: 1, or amino acidresidues on a TREM2 protein corresponding to amino acid residues 43-50of SEQ ID NO: 1. In certain embodiments, the isolated antibody binds toone or more amino acids within amino acid residues 49-57 of SEQ ID NO:1, or amino acid residues on a TREM2 protein corresponding to amino acidresidues 49-57 of SEQ ID NO: 1. In certain embodiments, the isolatedantibody binds to one or more amino acids within amino acid residues139-146 of SEQ ID NO: 1, or amino acid residues on a TREM2 proteincorresponding to amino acid residues 49-57 of SEQ ID NO: 1. In certainembodiments, the isolated antibody binds to one or more amino acidswithin amino acid residues 140-153 of SEQ ID NO: 1, or amino acidresidues on a TREM2 protein corresponding to amino acid residues 140-153of SEQ ID NO: 1. In certain embodiments, the isolated antibody binds toan epitope comprising one or more amino acids within amino acid residues43-50 of SEQ ID NO: 1. In certain embodiments, the isolated antibodybinds to an epitope comprising one or more amino acids within amino acidresidues 49-57 of SEQ ID NO: 1. In certain embodiments, the isolatedantibody binds to an epitope comprising one or more amino acids withinamino acid residues 139-146 of SEQ ID NO: 1. In certain embodiments, theisolated antibody binds to an epitope comprising one or more amino acidswithin amino acid residues 140-153 of SEQ ID NO: 1.

In certain embodiments that may be combined with any of the precedingembodiments, the TREM2 protein is a mammalian protein or a humanprotein. In certain embodiments that may be combined with any of thepreceding embodiments, the TREM2 protein is a wild-type protein. Incertain embodiments that may be combined with any of the precedingembodiments, the TREM2 protein is a naturally occurring variant. Incertain embodiments that may be combined with any of the precedingembodiments, the antibody is an agonist antibody, and wherein theantibody induces one or more TREM2 activities. In certain embodimentsthat may be combined with any of the preceding embodiments, the isolatedantibody induces or retains TREM2 clustering on a cell surface. Incertain embodiments that may be combined with any of the precedingembodiments, the one or more TREM2 activities are selected from thegroup consisting of: i. TREM2 binding to DAP12; ii. DAP12phosphorylation; iii. increasing the survival of macrophages, microglialcells, M1 microglial cells, activated M1 microglial cells, M2 microglialcells, dendritic cells, macrophages, M1 macrophages, activated M1macrophages, M2 macrophages, monocytes, osteoclasts, Langerhans cells ofskin, and/or Kupffer cells; iv. Syk phosphorylation; v. increasedexpression of CD83 and/or CD86 on dendritic cells; vi. increasingphagocytosis by macrophages, dendritic cells, monocytes, and/ormicroglia; vii. increasing activity of one or more TREM2-dependentgenes, optionally wherein the one or more TREM2-dependent genes compriseone or more nuclear factor of activated T-cells (NFAT) transcriptionfactors; and viii. increasing expression of one or more mediatorsselected from the group consisting of IL-12p70, IL-6, and IL-10. Incertain embodiments that may be combined with any of the precedingembodiments, the antibody is of the IgG class the IgM class, or the IgAclass. In certain embodiments that may be combined with any of thepreceding embodiments, the antibody is of the IgG class and has an IgG1,IgG2, IgG3, or IgG4 isotype. In certain embodiments that may be combinedwith any of the preceding embodiments, the antibody has an IgG2 isotype.In certain embodiments that may be combined with any of the precedingembodiments, the antibody comprises a human IgG2 constant region. Incertain embodiments that may be combined with any of the precedingembodiments, the human IgG2 constant region comprises an Fc region. Incertain embodiments that may be combined with any of the precedingembodiments, the antibody induces the one or more TREM2 activitiesindependently of binding to an Fc receptor. In certain embodiments thatmay be combined with any of the preceding embodiments, the antibodybinds an inhibitory Fc receptor. In certain embodiments that may becombined with any of the preceding embodiments, the inhibitory Fcreceptor is inhibitory Fc-gamma receptor IIB (FcγIIB). In certainembodiments that may be combined with any of the preceding embodiments:i. the isolated antibody has a human IgG2 isotype and comprises one ormore amino acid substitutions in the Fc region at a residue positionselected from the group consisting of V234A, G237A, H268Q, V309L, A330S,P331S, C232S, C233S, S267E, L328F, M252Y, S254T, T256E, and anycombination thereof, wherein the numbering of the residues is accordingto EU or Kabat numbering; ii. the isolated antibody has a human IgG2isotype, wherein the human IgG2 comprises a constant region, and whereinthe human IgG2 constant region comprises a light chain constant regioncomprising a C214S amino acid substitution, wherein the numbering of theresidues is according to EU or Kabat numbering; iii. the isolatedantibody has a human or mouse IgG1 isotype and comprises one or moreamino acid substitutions in the Fc region at a residue position selectedfrom the group consisting of N297A, D265A, L234A, L235A, G237A, C226S,C229S, E233P, L234V, L234F, L235E, P331S, S267E, L328F, A330L, M252Y,S254T, T256E, and any combination thereof, wherein the numbering of theresidues is according to EU or Kabat numbering; iv. the isolatedantibody has an IgG1 isotype and comprises an IgG2 isotype heavy chainconstant domain 1(CH1) and hinge region, optionally wherein the IgG2isotype CH1 and hinge region comprise the amino acid sequence ofASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSNFGTQT YTCNVDHKPS NTKVDKTVERKCCVECPPCP (SEQ ID NO: 397),and optionally wherein the antibody Fc region comprises a S267E aminoacid substitution, a L328F amino acid substitution, or both, and/or aN297A or N297Q amino acid substitution, wherein the numbering of theresidues is according to EU or Kabat numbering; v. the isolated antibodyhas a human or mouse IgG4 isotype and comprises one or more amino acidsubstitutions in the Fc region at a residue position selected from thegroup consisting of L235A, G237A, S228P, L236E, S267E, E318A, L328F,M252Y, S254T, T256E, and any combination thereof, wherein the numberingof the residues is according to EU or Kabat numbering; or vi. theisolated antibody has a hybrid IgG2/4 isotype, optionally wherein theantibody comprises an amino acid sequence comprising amino acids 118 to260 of human IgG2 and amino acids 261 to 447 of human IgG4, wherein thenumbering of the residues is according to EU or, Kabat numbering. Incertain embodiments that may be combined with any of the precedingembodiments, the isolated antibody is an inert antibody. In certainembodiments that may be combined with any of the preceding embodiments,the isolated antibody is an antagonist antibody. In certain embodimentsthat may be combined with any of the preceding embodiments, the isolatedantibody inhibits one or more TREM2 activities. In certain embodimentsthat may be combined with any of the preceding embodiments, theinhibited one or more TREM2 activities are selected from the groupconsisting of decreasing activity of one or more TREM2-dependent genes;decreasing activity of one or more nuclear factor of activated T-cells(NFAT) transcription factors; decreasing the survival of macrophages,microglial cells, M1 macrophages, M1 microglial cells, M2 macrophages,M2 microglial cells, osteoclasts, Langerhans cells of skin, Kupffercells, and/or dendritic cells; decreased expression of one or moremediators selected from the group consisting of IL-12p70, IL-6, andIL-10; and any combination thereof. In certain embodiments that may becombined with any of the preceding embodiments, the isolated antibodyinhibits interaction between TREM2 and one or more TREM2 ligands,inhibits TREM2 signal transduction, or both. In certain embodiments thatmay be combined with any of the preceding embodiments, the antibody isincapable of binding an Fc-gamma receptor (FcγR). In certain embodimentsthat may be combined with any of the preceding embodiments: i. theisolated antibody has a human or mouse IgG1 isotype and comprises one ormore amino acid substitutions in the Fc region are at a residue positionselected from the group consisting of N297A, N297Q, D265A, L234A, L235A,C226S, C229S, P238S, E233P, L234V, P238A, A327Q, A327G, P329A, K322A,L234F, L235E, P331S, T394D, A330L, M252Y, S254T, T256E, and anycombination thereof, wherein the numbering of the residues is accordingto EU or Kabat numbering, optionally wherein the Fc region furthercomprises an amino acid deletion at a position corresponding to glycine236 according to EU or Kabat numbering; ii. the isolated antibody has ahuman IgG2 isotype and comprises one or more amino acid substitutions inthe Fc region are at a residue position selected from the groupconsisting of V234A, G237A, H268E, V309L, N297A, N297Q, A330S, P331S,C232S, C233S, M252Y, S254T, T256E, and any combination thereof, whereinthe numbering of the residues is according to EU or Kabat numbering; oriii. the isolated antibody has a human or mouse IgG4 isotype andcomprises one or more amino acid substitutions in the Fc region are at aresidue position selected from the group consisting of E233P, F234V,L235A, G237A, E318A, S228P, L236E, S241P, L248E, T394D, M252Y, S254T,T256E, N297A, N297Q, and any combination thereof, wherein the numberingof the residues is according to EU or Kabat numbering. In certainembodiments that may be combined with any of the preceding embodiments,the Fc region further comprises one or more additional amino acidsubstitutions at a position selected from the group consisting of A330L,L234F; L235E, P331S, and any combination thereof, wherein the numberingof the residues is according to EU or Kabat numbering. In certainembodiments that may be combined with any of the preceding embodiments,the Fc region further comprises a S228P amino acid substitutionaccording to EU or Kabat numbering. In certain embodiments that may becombined with any of the preceding embodiments, the Fc region furthercomprises one or more additional amino acid substitutions at a positionselected from the group consisting of M252Y, S254T, T256E, and anycombination thereof, wherein the numbering of the residues is accordingto EU or Kabat numbering. In certain embodiments that may be combinedwith any of the preceding embodiments, the isolated antibody is anantibody fragment that binds to one or more human proteins selected fromthe group consisting of human TREM2, and a naturally occurring variantof human TREM2, and wherein the antibody fragment is cross-linked to asecond antibody fragment that binds to one or more human proteinsselected from the group consisting of human TREM2, a naturally occurringvariant of human TREM2, human DAP12, and naturally occurring variant ofhuman DAP12. In certain embodiments that may be combined with any of thepreceding embodiments, the isolated antibody is an antibody fragment,and wherein the antibody fragment is an Fab, Fab′, Fab′-SH, F(ab′)2, Fvor scFv fragment. In certain embodiments that may be combined with anyof the preceding embodiments, the isolated antibody competes for bindingof TREM2 with one or more TREM2 ligands. In certain embodiments that maybe combined with any of the preceding embodiments, the one or more TREM2ligands are selected from the group consisting of E. coli cells,apoptotic cells, nucleic acids, anionic lipids, zwitterionic lipids,negatively charged phospholipids, phosphatidylserine, sulfatides,phosphatidylcholin, sphingomyelin, membrane phospholipids, lipidatedproteins, proteolipids, lipidated peptides, and lipidated amyloid betapeptide. In certain embodiments that may be combined with any of thepreceding embodiments, the isolated antibody is a human antibody, ahumanized antibody, a bispecific antibody, a multivalent antibody, or achimeric antibody. In certain embodiments that may be combined with anyof the preceding embodiments, the isolated antibody is a bispecificantibody recognizing a first antigen and a second antigen. In certainembodiments that may be combined with any of the preceding embodiments,the first antigen is human TREM2 or a naturally occurring variantthereof, and the second antigen is a disease-causing protein selectedfrom the group consisting of amyloid beta or fragments thereof, Tau,IAPP, alpha-synuclein, TDP-43, FUS protein, prion protein, PrPSc,huntingtin, calcitonin, superoxide dismutase, ataxin, Lewy body, atrialnatriuretic factor, islet amyloid polypeptide, insulin, apolipoproteinAI, serum amyloid A, medin, prolactin, transthyretin, lysozyme, beta 2microglobulin, gelsolin, keratoepithelin, cystatin, immunoglobulin lightchain AL, S-IBM protein, Repeat-associated non-ATG (RAN) translationproducts, DiPeptide repeat (DPR) peptides, glycine-alanine (GA) repeatpeptides, glycine-proline (GP) repeat peptides, glycine-arginine (GR)repeat peptides, proline-alanine (PA) repeat peptides, andproline-arginine (PR) repeat peptides; or a blood brain barriertargeting protein selected from the group consisting of: trasnferinreceptor, insulin receptor, insulin like growth factor receptor, LRP-1,and LRP1. In certain embodiments that may be combined with any of thepreceding embodiments, the isolated antibody is an antibody fragmentthat binds to one or more human proteins selected from the groupconsisting of human TREM2, and a naturally occurring variant of humanTREM2; and wherein the antibody is used in combination with one or moreantibodies that specifically bind a disease-causing protein selectedfrom the group consisting of: amyloid beta or fragments thereof, Tau,IAPP, alpha-synuclein, TDP-43, FUS protein, prion protein, PrPSc,huntingtin, calcitonin, superoxide dismutase, ataxin, Lewy body, atrialnatriuretic factor, islet amyloid polypeptide, insulin, apolipoproteinAI, serum amyloid A, medin, prolactin, transthyretin, lysozyme, beta 2microglobulin, gelsolin, keratoepithelin, cystatin, immunoglobulin lightchain AL, S-IBM protein, Repeat-associated non-ATG (RAN) translationproducts, DiPeptide repeat (DPR) peptides, glycine-alanine (GA) repeatpeptides, glycine-proline (GP) repeat peptides, glycine-arginine (GR)repeat peptides, proline-alanine (PA) repeat peptides, andproline-arginine (PR) repeat peptides, and any combination thereof. Incertain embodiments that may be combined with any of the precedingembodiments, the antibody is a monoclonal antibody. In certainembodiments that may be combined with any of the preceding embodiments,the isolated antibody is a bispecific antibody that binds to TREM2 andDAP12.

In certain embodiments that may be combined with any of the precedingembodiments, the isolated antibody comprises a heavy chain variabledomain and a light chain variable domain, wherein the heavy chainvariable domain comprises: (a) an HVR-H1 comprising an amino acidsequence selected from the group consisting of SEQ ID NOs:3-24, 398, and404; an HVR-H2 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs:25-49, 399, and 405; and (c) an HVR-H3 ccomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs:50-119, 400, and 406; and/or wherein the light chain variabledomain comprises: (a) an HVR-L1 c comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs:120-137, 401, and 407;(b) an HVR-L2 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs:138-152, 402, and 408; and (c) an HVR-L3comprising an amino acid sequence selected from the group consisting ofSEQ ID NOs:153-236, 403, and 409.

Other aspects of the present disclosure relate to an isolated anti-humanTREM2 antibody, wherein the isolated antibody comprises a heavy chainvariable domain and a light chain variable domain, wherein the heavychain variable domain comprises: (a) an HVR-H1 comprising an amino acidsequence selected from the group consisting of SEQ ID NOs:3-24, 398, and404; an HVR-H2 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs:25-49, 399, and 405; and (c) an HVR-H3 ccomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs:50-119, 400, and 406; and/or wherein the light chain variabledomain comprises: (a) an HVR-L1 c comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs:120-137, 401, and 407;(b) an HVR-L2 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs:138-152, 402, and 408; and (c) an HVR-L3comprising an amino acid sequence selected from the group consisting ofSEQ ID NOs:153-236, 403, and 409. Other aspects of the presentdisclosure relate to an isolated anti-human TREM2 antibody which bindsessentially the same TREM2 epitope as a monoclonal antibody selectedfrom the group consisting of: Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8,Ab9, Ab10, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19, Ab20,Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32,Ab33, Ab34, Ab35, Ab36, Ab37, Ab38, Ab39, Ab40, Ab41, Ab42, Ab43, Ab44,Ab45, Ab46, Ab47, Ab48, Ab49, Ab50, Ab51, Ab52, Ab53, Ab54, Ab55, Ab56,Ab57, Ab58, Ab59, Ab60, Ab61, Ab62, Ab63, Ab64, Ab65, Ab66, Ab67, Ab68,Ab69, Ab70, Ab71, Ab72, Ab73, Ab74, Ab75, Ab76, Ab77, Ab78, Ab79, Ab80,Ab81, Ab82, Ab83, Ab84, Ab85, Ab86, and Ab87. Other aspects of thepresent disclosure relate to an isolated anti-human TREM2 antibody whichcompetes with a monoclonal antibody selected from the group consistingof: Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab12, Ab13,Ab14, Ab15, Ab16, Ab17, Ab18, Ab19, Ab20, Ab21, Ab22, Ab23, Ab24, Ab25,Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, Ab34, Ab35, Ab36, Ab37,Ab38, Ab39, Ab40, Ab41, Ab42, Ab43, Ab44, Ab45, Ab46, Ab47, Ab48, Ab49,Ab50, Ab51, Ab52, Ab53, Ab54, Ab55, Ab56, Ab57, Ab58, Ab59, Ab60, Ab61,Ab62, Ab63, Ab64, Ab65, Ab66, Ab67, Ab68, Ab69, Ab70, Ab71, Ab72, Ab73,Ab74, Ab75, Ab76, Ab77, Ab78, Ab79, Ab80, Ab81, Ab82, Ab83, Ab84, Ab85,Ab86, and Ab87 for binding to TREM2.

In certain embodiments that may be combined with any of the precedingembodiments, the antibody is an agonist antibody, and wherein theantibody induces one or more TREM2 activities. In certain embodimentsthat may be combined with any of the preceding embodiments, the isolatedantibody induces or retains TREM2 clustering on a cell surface. Incertain embodiments that may be combined with any of the precedingembodiments, the one or more TREM2 activities are selected from thegroup consisting of TREM2 binding to DAP12; DAP12 phosphorylation;increasing the survival of macrophages, microglial cells, M1 microglialcells, activated M1 microglial cells, M2 microglial cells, dendriticcells, macrophages, M1 macrophages, activated M1 macrophages, M2macrophages, monocytes, osteoclasts, Langerhans cells of skin, and/orKupffer cells; increased expression of IL-6; Syk phosphorylation;increased expression of CD83 and/or CD86 on dendritic cells; increasingphagocytosis by macrophages, dendritic cells, monocytes, and/ormicroglia; and increasing activity of one or more TREM2-dependent genes,optionally wherein the one or more TREM2-dependent genes comprise one ormore nuclear factor of activated T-cells (NFAT) transcription factors;and any combination thereof. In certain embodiments that may be combinedwith any of the preceding embodiments, the antibody is of the IgG classthe IgM class, or the IgA class. In certain embodiments that may becombined with any of the preceding embodiments, the antibody is of theIgG class and has an IgG1, IgG2, IgG3, or IgG4 isotype. In certainembodiments that may be combined with any of the preceding embodiments,the antibody has an IgG2 isotype. In certain embodiments that may becombined with any of the preceding embodiments, the antibody comprises ahuman IgG2 constant region. In certain embodiments that may be combinedwith any of the preceding embodiments, the human IgG2 constant regioncomprises an Fc region. In certain embodiments that may be combined withany of the preceding embodiments, the antibody induces the one or moreTREM2 activities independently of binding to an Fc receptor. In certainembodiments that may be combined with any of the preceding embodiments,the antibody binds an inhibitory Fc receptor. In certain embodimentsthat may be combined with any of the preceding embodiments, theinhibitory Fc receptor is inhibitory Fc-gamma receptor IIB (FcγIIB). Incertain embodiments that may be combined with any of the precedingembodiments: i. the isolated antibody has a human IgG2 isotype andcomprises one or more amino acid substitutions in the Fc region at aresidue position selected from the group consisting of V234A, G237A,H268Q, V309L, A330S, P331S, C232S, C233S, S267E, L328F, M252Y, S254T,T256E, and any combination thereof, wherein the numbering of theresidues is according to EU or Kabat numbering; ii. the isolatedantibody has a human IgG2 isotype, wherein the human IgG2 comprises aconstant region, and wherein the human IgG2 constant region comprises alight chain constant region comprising a C214S amino acid substitution,wherein the numbering of the residues is according to EU or Kabatnumbering; iii. the isolated antibody has a human or mouse IgG1 isotypeand comprises one or more amino acid substitutions in the Fc region at aresidue position selected from the group consisting of N297A, D265A,L234A, L235A, G237A, C226S, C229S, E233P, L234V, L234F, L235E, P331S,S267E, L328F, A330L, M252Y, S254T, T256E, and any combination thereof,wherein the numbering of the residues is according to EU or Kabatnumbering; iv. the isolated antibody has an IgG1 isotype and comprisesan IgG2 isotype heavy chain constant domain 1(CH1) and hinge region,optionally wherein the IgG2 isotype CH1 and hinge region comprise theamino acid sequence of ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVT VPSSNFGTQT YTCNVDHKPSNTKVDKTVERKCCVECPPCP (SEQ ID NO: 397), and optionally wherein theantibody Fc region comprises a S267E amino acid substitution, a L328Famino acid substitution, or both, and/or a N297A or N297Q amino acidsubstitution, wherein the numbering of the residues is according to EUor Kabat numbering; v. the isolated antibody has a human or mouse IgG1isotype and comprises one or more amino acid substitutions in the Fcregion at a residue position selected from the group consisting ofL235A, G237A, S228P, L236E, S267E, E318A, L328F, M252Y, S254T, T256E,and any combination thereof, wherein the numbering of the residues isaccording to EU or Kabat numbering; or vi. the isolated antibody has ahybrid IgG2/4 isotype, optionally wherein the antibody comprises anamino acid sequence comprising amino acids 118 to 260 of human IgG2 andamino acids 261 to 447 of human IgG4, wherein the numbering of theresidues is according to EU or, Kabat numbering. In certain embodimentsthat may be combined with any of the preceding embodiments, the isolatedantibody is an antibody fragment that binds to one or more humanproteins selected from the group consisting of human TREM2, and anaturally occurring variant of human TREM2, and wherein the antibodyfragment is cross-linked to a second antibody fragment that binds to oneor more human proteins selected from the group consisting of humanTREM2, a naturally occurring variant of human TREM2, human DAP12, andnaturally occurring variant of human DAP12. In certain embodiments thatmay be combined with any of the preceding embodiments, the isolatedantibody is an antibody fragment, and wherein the antibody fragment isan Fab, Fab′, Fab′-SH, F(ab′)2, Fv or scFv fragment. In certainembodiments that may be combined with any of the preceding embodiments,the isolated antibody is an inert antibody. In certain embodiments thatmay be combined with any of the preceding embodiments, the isolatedantibody is an antagonist antibody. In certain embodiments that may becombined with any of the preceding embodiments, the isolated antibodyinhibits one or more TREM2 activities. In certain embodiments that maybe combined with any of the preceding embodiments, the inhibited one ormore TREM2 activities are selected from the group consisting ofdecreasing activity of one or more TREM2-dependent genes; decreasingactivity of one or more nuclear factor of activated T-cells (NFAT)transcription factors; decreasing the survival of macrophages,microglial cells, monocytes, osteoclasts, Langerhans cells of skin,Kupffer cells, and/or dendritic cells; decreased expression of one ormore mediators selected from the group consisting of IL-12p70, IL-6, andIL-10; and any combination thereof. In certain embodiments that may becombined with any of the preceding embodiments, the isolated antibodyinhibits interaction between TREM2 and one or more TREM2 ligands,inhibits TREM2 signal transduction, or both. In certain embodiments thatmay be combined with any of the preceding embodiments, the antibody isincapable of binding an Fc-gamma receptor (FcγR). In certain embodimentsthat may be combined with any of the preceding embodiments: i. theisolated antibody has a human or mouse IgG1 isotype and comprises one ormore amino acid substitutions in the Fc region are at a residue positionselected from the group consisting of N297A, N297Q, D265A, L234A, L235A,C226S, C229S, P238S, E233P, L234V, P238A, A327Q, A327G, P329A, K322A,L234F, L235E, P331S, T394D, A330L, M252Y, S254T, T256E, and anycombination thereof, wherein the numbering of the residues is accordingto EU or Kabat numbering, optionally wherein the Fc region furthercomprises an amino acid deletion at a position corresponding to glycine236 according to EU or Kabat numbering; ii. the isolated antibody has ahuman IgG2 isotype and comprises one or more amino acid substitutions inthe Fc region are at a residue position selected from the groupconsisting of V234A, G237A, H268E, V309L, N297A, N297Q, A330S, P331S,C232S, C233S, M252Y, S254T, T256E, and any combination thereof, whereinthe numbering of the residues is according to EU or Kabat numbering; oriii. the isolated antibody has a human or mouse IgG4 isotype andcomprises one or more amino acid substitutions in the Fc region are at aresidue position selected from the group consisting of E233P, F234V,L235A, G237A, E318A, S228P, L236E, S241P, L248E, T394D, M252Y, S254T,T256E, N297A, N297Q, and any combination thereof, wherein the numberingof the residues is according to EU or Kabat numbering. In certainembodiments that may be combined with any of the preceding embodiments,the isolated antibody is an antibody fragment that binds to one or morehuman proteins selected from the group consisting of human TREM2, and anaturally occurring variant of human TREM2. In certain embodiments thatmay be combined with any of the preceding embodiments, the isolatedantibody is an antibody fragment, and wherein the antibody fragment isan Fab, Fab′, Fab′-SH, F(ab′)2, Fv or scFv fragment. In certainembodiments that may be combined with any of the preceding embodiments,the Fc region further comprises one or more additional amino acidsubstitutions at a position selected from the group consisting of A330L,L234F; L235E, P331S, and any combination thereof, wherein the numberingof the residues is according to EU or Kabat numbering. In certainembodiments that may be combined with any of the preceding embodiments,the Fc region further comprises a S228P amino acid substitutionaccording to EU or Kabat numbering. In certain embodiments that may becombined with any of the preceding embodiments, the Fc region furthercomprises one or more additional amino acid substitutions at a positionselected from the group consisting of M252Y, S254T, T256E, and anycombination thereof, wherein the numbering of the residues is accordingto EU or Kabat numbering. In certain embodiments that may be combinedwith any of the preceding embodiments, the antibody is a human antibody,a humanized antibody, a bispecific antibody, a multivalent antibody, ora chimeric antibody. In certain embodiments that may be combined withany of the preceding embodiments, the antibody is a bispecific antibodyrecognizing a first antigen and a second antigen. In certain embodimentsthat may be combined with any of the preceding embodiments, the antibodyis a monoclonal antibody.

In certain embodiments that may be combined with any of the precedingembodiments, the isolated antibody binds specifically to both humanTREM2 and mouse TREM2. In certain embodiments that may be combined withany of the preceding embodiments, the isolated antibody has dissociationconstant (K_(D)) for human TREM2 and mouse TREM2 that ranges from lessthan about 5.75 nM to less than about 0.09 nM. In certain embodimentsthat may be combined with any of the preceding embodiments, the isolatedantibody has dissociation constant (K_(D)) for human TREM2-Fc fusionprotein that ranges from less than about 1.51 nM to less than about 0.35nM. In certain embodiments that may be combined with any of thepreceding embodiments, the isolated antibody has dissociation constant(K_(D)) for human monomeric TREM2 protein that ranges from less thanabout 5.75 nM to less than about 1.15 nm. In certain embodiments thatmay be combined with any of the preceding embodiments, the isolatedantibody has dissociation constant (K_(D)) for mouse TREM2-Fc fusionprotein that ranges from less than about 0.23 nM to less than about 0.09nM. In certain embodiments that may be combined with any of thepreceding embodiments, the isolated antibody has dissociation constant(K_(D)) for human TREM2 and mouse TREM2 that ranges from less than about6.70 nM to less than about 0.23 nM. In certain embodiments that may becombined with any of the preceding embodiments, the isolated antibodyhas dissociation constant (K_(D)) for human TREM2-Fc fusion protein thatranges from less than about 0.71 nM to less than about 0.23 nM. Incertain embodiments that may be combined with any of the precedingembodiments, the isolated antibody has dissociation constant (K_(D)) forhuman monomeric TREM2 protein that ranges from less than about 6.70 nMto less than about 0.66 nM. In certain embodiments that may be combinedwith any of the preceding embodiments, the isolated antibody hasdissociation constant (K_(D)) for mouse TREM2-Fc fusion protein thatranges from less than about 4.90 nM to less than about 0.35 nM.

Other aspects of the present disclosure relate to an isolated nucleicacid encoding the antibody of any one of the preceding embodiments.Other aspects of the present disclosure relate to a vector comprisingthe nucleic acid of any one of the preceding embodiments. Other aspectsof the present disclosure relate to a host cell comprising the vector ofany one of the preceding embodiments. Other aspects of the presentdisclosure relate to an isolated host cell comprising the vector of anyone of the preceding embodiments. Other aspects of the presentdisclosure relate to a method of producing an antibody, comprisingculturing the cell of any one of the preceding embodiments so that theantibody is produced. In certain embodiments, the method furthercomprises recovering the antibody produced by the cell. Other aspects ofthe present disclosure relate to an isolated antibody produced by any ofthe preceding methods of producing an antibody. Other aspects of thepresent disclosure relate to a pharmaceutical composition comprising theantibody of any one of the preceding embodiments and a pharmaceuticallyacceptable carrier.

Other aspects of the present disclosure relate to a method ofpreventing, reducing risk, or treating an individual having a disease,disorder, or injury selected from the group consisting of dementia,frontotemporal dementia, Alzheimer's disease, Nasu-Hakola disease, andmultiple sclerosis, comprising administering to the individual atherapeutically effective amount of an isolated agonist antibody of anyone of the preceding embodiments. Other aspects of the presentdisclosure relate to an isolated agonist antibody of any one of thepreceding embodiments for use in preventing, reducing risk, or treatingan individual having a disease, disorder, or injury selected from thegroup consisting of dementia, frontotemporal dementia, Alzheimer'sdisease, Nasu-Hakola disease, and multiple sclerosis. Other aspects ofthe present disclosure relate to use of an isolated agonist antibody ofany one of the preceding embodiments in the manufacture of a medicamentfor preventing, reducing risk, or treating an individual having adisease, disorder, or injury selected from the group consisting ofdementia, frontotemporal dementia, Alzheimer's disease, Nasu-Hakoladisease, and multiple sclerosis. In certain embodiments that may becombined with any of the preceding embodiments, the individual has aheterozygous variant of TREM2, wherein the variant comprises one or moresubstitutions selected from the group consisting of: i. a glutamic acidto stop codon substitution in the nucleic acid sequence encoding aminoacid residue Glu14 of SEQ ID NO: 1; ii. a glutamine to stop codonsubstitution in the nucleic acid sequence encoding amino acid residueGln33 of SEQ ID NO: 1; iii. a tryptophan to stop codon substitution inthe nucleic acid sequence encoding amino acid residue Trp44 of SEQ IDNO: 1; iv. an arginine to histidine amino acid substitution at an aminoacid corresponding to amino acid residue Arg47 of SEQ ID NO: 1; v. atryptophan to stop codon substitution in the nucleic acid sequenceencoding amino acid residue Trp78 of SEQ ID NO: 1; vi. a valine toglycine amino acid substitution at an amino acid corresponding to aminoacid residue Val126 of SEQ ID NO: 1; vii. an aspartic acid to glycineamino acid substitution at an amino acid corresponding to amino acidresidue Asp134 of SEQ ID NO: 1; and viii. a lysine to asparagine aminoacid substitution at an amino acid corresponding to amino acid residueLys186 of SEQ ID NO: 1. In certain embodiments that may be combined withany of the preceding embodiments, the individual has a heterozygousvariant of TREM2, wherein the variant comprises a guanine nucleotidedeletion at a nucleotide corresponding to nucleotide residue G313 of thenucleic acid sequence encoding SEQ ID NO: 1; a guanine nucleotidedeletion at a nucleotide corresponding to nucleotide residue G267 of thenucleic acid sequence encoding SEQ ID NO: 1; or both. In certainembodiments that may be combined with any of the preceding embodiments,the individual has a heterozygous variant of DAP12, wherein the variantcomprises one or more variants selected from the group consisting of: i.a methionine to threonine substitution at an amino acid corresponding toamino acid residue Met1 of SEQ ID NO: 2; ii. a glycine to arginine aminoacid substitution at an amino acid corresponding to amino acid residueGly49 of SEQ ID NO: 2; iii. a deletion within exons 1-4 of the nucleicacid sequence encoding SEQ ID NO: 2; iv. an insertion of 14 amino acidresidues at exon 3 of the nucleic acid sequence encoding SEQ ID NO: 2;and v. a guanine nucleotide deletion at a nucleotide corresponding tonucleotide residue G141 of the nucleic acid sequence encoding SEQ ID NO:2.

Other aspects of the present disclosure relate to a method of inducingor promoting innate immune cell survival in an individual in needthereof, comprising administering to the individual a therapeuticallyeffective amount of an isolated agonist antibody of any one of thepreceding embodiments. Other aspects of the present disclosure relate toan isolated agonist antibody of any one of the preceding embodiments foruse in inducing or promoting innate immune cell survival in anindividual in need thereof. Other aspects of the present disclosurerelate to use of an isolated agonist antibody of any one of thepreceding embodiments in the manufacture of a medicament for inducing orpromoting innate immune cell survival in an individual in need thereof.Other aspects of the present disclosure relate to a method of inducingor promoting wound healing in an individual in need thereof, comprisingadministering to the individual a therapeutically effective amount of anisolated agonist antibody that binds to a TREM2 protein. Other aspectsof the present disclosure relate to an isolated agonist antibody thatbinds to a TREM2 protein for use in inducing or promoting wound healingin an individual in need thereof. Other aspects of the presentdisclosure relate to use of an isolated agonist antibody that binds to aTREM2 protein in the manufacture of a medicament for inducing orpromoting wound healing in an individual in need thereof.

Other aspects of the present disclosure relate to a method of decreasinginnate immune cell survival in an individual in need thereof, comprisingadministering to the individual a therapeutically effective amount of anisolated antagonist antibody of any one of the preceding embodiments.Other aspects of the present disclosure relate to an isolated antagonistantibody of any one of the preceding embodiments for use in decreasinginnate immune cell survival in an individual in need thereof. Otheraspects of the present disclosure relate to use of an isolatedantagonist antibody of any one of the preceding embodiments in themanufacture of a medicament for decreasing innate immune cell survivalin an individual in need thereof.

Other aspects of the present disclosure relate to a method ofpreventing, reducing risk, or treating an individual having a disease,disorder, or injury selected from the group consisting of dementia,frontotemporal dementia, Alzheimer's disease, vascular dementia, mixeddementia, Creutzfeldt-Jakob disease, normal pressure hydrocephalus,amyotrophic lateral sclerosis, Huntington's disease, Taupathy disease,Nasu-Hakola disease, stroke, acute trauma, chronic trauma, lupus, acuteand chronic colitis, wound healing, Crohn's disease, inflammatory boweldisease, ulcerative colitis, obesity, Malaria, essential tremor, centralnervous system lupus, Behcet's disease, Parkinson's disease, dementiawith Lewy bodies, multiple system atrophy, Shy-Drager syndrome,progressive supranuclear palsy, cortical basal ganglionic degeneration,acute disseminated encephalomyelitis, granulomartous disorders,Sarcoidosis, diseases of aging, seizures, spinal cord injury, traumaticbrain injury, age related macular degeneration, glaucoma, retinitispigmentosa, retinal degeneration, respiratory tract infection, sepsis,eye infection, systemic infection, lupus, arthritis, multiple sclerosis,low bone density, osteoporosis, osteogenesis, osteopetrotic disease,Paget's disease of bone, and cancer, comprising administering to theindividual a therapeutically effective amount of an isolated antibody ofany one of the preceding embodiments. Other aspects of the presentdisclosure relate to an isolated antibody of any one of the precedingembodiments for use in preventing, reducing risk, or treating anindividual having a disease, disorder, or injury selected from the groupconsisting of dementia, frontotemporal dementia, Alzheimer's disease,vascular dementia, mixed dementia, Creutzfeldt-Jakob disease, normalpressure hydrocephalus, amyotrophic lateral sclerosis, Huntington'sdisease, Taupathy disease, Nasu-Hakola disease, stroke, acute trauma,chronic trauma, lupus, acute and chronic colitis, wound healing, Crohn'sdisease, inflammatory bowel disease, ulcerative colitis, obesity,Malaria, essential tremor, central nervous system lupus, Behcet'sdisease, Parkinson's disease, dementia with Lewy bodies, multiple systematrophy, Shy-Drager syndrome, progressive supranuclear palsy, corticalbasal ganglionic degeneration, acute disseminated encephalomyelitis,granulomartous disorders, Sarcoidosis, diseases of aging, seizures,spinal cord injury, traumatic brain injury, age related maculardegeneration, glaucoma, retinitis pigmentosa, retinal degeneration,respiratory tract infection, sepsis, eye infection, systemic infection,lupus, arthritis, multiple sclerosis, low bone density, osteoporosis,osteogenesis, osteopetrotic disease, Paget's disease of bone, andcancer. Other aspects of the present disclosure relate to use of anisolated antibody of any one of the preceding embodiments in themanufacture of a medicament for preventing, reducing risk, or treatingan individual having a disease, disorder, or injury selected from thegroup consisting of dementia, frontotemporal dementia, Alzheimer'sdisease, vascular dementia, mixed dementia, Creutzfeldt-Jakob disease,normal pressure hydrocephalus, amyotrophic lateral sclerosis,Huntington's disease, Taupathy disease, Nasu-Hakola disease, stroke,acute trauma, chronic trauma, lupus, acute and chronic colitis, woundhealing, Crohn's disease, inflammatory bowel disease, ulcerativecolitis, obesity, Malaria, essential tremor, central nervous systemlupus, Behcet's disease, Parkinson's disease, dementia with Lewy bodies,multiple system atrophy, Shy-Drager syndrome, progressive supranuclearpalsy, cortical basal ganglionic degeneration, acute disseminatedencephalomyelitis, granulomartous disorders, Sarcoidosis, diseases ofaging, seizures, spinal cord injury, traumatic brain injury, age relatedmacular degeneration, glaucoma, retinitis pigmentosa, retinaldegeneration, respiratory tract infection, sepsis, eye infection,systemic infection, lupus, arthritis, multiple sclerosis, low bonedensity, osteoporosis, osteogenesis, osteopetrotic disease, Paget'sdisease of bone, and cancer.

In certain embodiments that may be combined with any of the precedingembodiments, the isolated antibody is: (a) an agonist antibody; (b) aninert antibody; or an (c) an antagonist antibody. In certain embodimentsthat may be combined with any of the preceding embodiments, (a) theantibody is of the IgG class the IgM class, or the IgA class; and/or (b)the antibody has an IgG1, IgG2, IgG3, or IgG4 isotype. In certainembodiments that may be combined with any of the preceding embodiments,the antibody comprises one or more amino acid substitutions in the Fcregion are at a residue position selected from the group consisting of:(a) V234A, G237A, H268Q, V309L, A330S, P331S, C232S, C233S, S267E,L328F, M252Y, S254T, T256E, and any combination thereof; (b) N297A,D265A, L234A, L235A, G237A, C226S, C229S, E233P, L234V, L234F, L235E,P331S, S267E, L328F, A330L, M252Y, S254T, T256E, and any combinationthereof; (c) L235A, G237A, S228P, L236E, S267E, E318A, L328F, M252Y,S254T, T256E, and any combination thereof; (d) N297A, N297Q, D265A,L234A, L235A, C226S, C229S, P238S, E233P, L234V, P238A, A327Q, A327G,P329A, K322A, L234F, L235E, P331S, T394D, A330L, M252Y, S254T, T256E,and any combination thereof; (e) V234A, G237A, H268E, V309L, N297A,N297Q, A330S, P331S, C232S, C233S, M252Y, S254T, T256E, and anycombination thereof; or (f) E233P, F234V, L235A, G237A, E318A, S228P,L236E, S241P, L248E, T394D, M252Y, S254T, T256E, N297A, N297Q, and anycombination thereof, wherein the numbering of the residues is accordingto EU or Kabat numbering. In certain embodiments that may be combinedwith any of the preceding embodiments, the isolated antibody: (a) bindsto one or more amino acids within amino acid residues 43-50 of SEQ IDNO: 1, or amino acid residues on a TREM2 protein corresponding to aminoacid residues 43-50 of SEQ ID NO: 1; or (b) one or more amino acidswithin amino acid residues 49-57 of SEQ ID NO: 1, or amino acid residueson a TREM2 protein corresponding to amino acid residues 49-57 of SEQ IDNO: 1. In certain embodiments that may be combined with any of thepreceding embodiments, the isolated antibody: (a) binds essentially thesame TREM2 epitope as the antibody Ab52; (b) comprises a heavy chainvariable domain and a light chain variable domain, wherein the heavychain variable domain comprises the HVR-H1, HVR-H2, and/or HVR-H3 of themonoclonal antibody Ab52; and/or wherein the light chain variable domaincomprises the HVR-L1, HVR-L2, and/or HVR-L3 of the monoclonal antibodyAb52; (c) comprises a heavy chain variable domain and a light chainvariable domain, wherein the heavy chain variable domain comprises anHVR-H1 comprising the amino acid sequence of SEQ ID NO:398, or an aminoacid sequence with at least about 95% homology to the amino acidsequence of SEQ ID NO:398, an HVR-H2 comprising the amino acid sequenceof SEQ ID NO:399, or an amino acid sequence with at least about 95%homology to the amino acid sequence of SEQ ID NO:399, and an HVR-H3comprising the amino acid sequence of SEQ ID NO:400, or an amino acidsequence with at least about 95% homology to the amino acid sequence ofSEQ ID NO:400, and/or wherein the light chain variable domain comprisesan HVR-L1 comprising the amino acid sequence of SEQ ID NO:401, or anamino acid sequence with at least about 95% homology to the amino acidsequence of SEQ ID NO:401, an HVR-L2 comprising the amino acid sequenceof SEQ ID NO:402, or an amino acid sequence with at least about 95%homology to the amino acid sequence of SEQ ID NO:402, and an HVR-L3comprising the amino acid sequence of SEQ ID NO:403, or an amino acidsequence with at least about 95% homology to the amino acid sequence ofSEQ ID NO:403; (d) binds essentially the same TREM2 epitope as theantibody Ab21; (e) comprises a heavy chain variable domain and a lightchain variable domain, wherein the heavy chain variable domain comprisesthe HVR-H1, HVR-H2, and/or HVR-H3 of the monoclonal antibody Ab21;and/or wherein the light chain variable domain comprises the HVR-L1,HVR-L2, and/or HVR-L3 of the monoclonal antibody Ab21; or (f) comprisesa heavy chain variable domain and a light chain variable domain, whereinthe heavy chain variable domain comprises an HVR-H1 comprising the aminoacid sequence of SEQ ID NO:404, or an amino acid sequence with at leastabout 95% homology to the amino acid sequence of SEQ ID NO:404, anHVR-H2 comprising the amino acid sequence of SEQ ID NO:405, or an aminoacid sequence with at least about 95% homology to the amino acidsequence of SEQ ID NO:405, and an HVR-H3 comprising the amino acidsequence of SEQ ID NO:406, or an amino acid sequence with at least about95% homology to the amino acid sequence of SEQ ID NO:406, and/or whereinthe light chain variable domain comprises an HVR-L1 comprising the aminoacid sequence of SEQ ID NO:407, or an amino acid sequence with at leastabout 95% homology to the amino acid sequence of SEQ ID NO:407, anHVR-L2 comprising the amino acid sequence of SEQ ID NO:408, or an aminoacid sequence with at least about 95% homology to the amino acidsequence of SEQ ID NO:408, and an HVR-L3 comprising the amino acidsequence of SEQ ID NO:409, or an amino acid sequence with at least about95% homology to the amino acid sequence of SEQ ID NO:409. In certainembodiments that may be combined with any of the preceding embodiments,the isolated antibody is the isolated antibody of any one of thepreceding embodiments. In certain embodiments that may be combined withany of the preceding embodiments, the isolated agonist antibody is theisolated agonist antibody of any one of the preceding embodiments. Incertain embodiments that may be combined with any of the precedingembodiments, the individual has a heterozygous variant of TREM2, whereinthe variant comprises one or more substitutions selected from the groupconsisting of: i. a glutamic acid to stop codon substitution in thenucleic acid sequence encoding amino acid residue Glu14 of SEQ ID NO: 1;ii. a glutamine to stop codon substitution in the nucleic acid sequenceencoding amino acid residue Gln33 of SEQ ID NO: 1; iii. a tryptophan tostop codon substitution in the nucleic acid sequence encoding amino acidresidue Trp44 of SEQ ID NO: 1; iv. an arginine to histidine amino acidsubstitution at an amino acid corresponding to amino acid residue Arg47of SEQ ID NO: 1; v. a tryptophan to stop codon substitution in thenucleic acid sequence encoding amino acid residue Trp78 of SEQ ID NO: 1;vi. a valine to glycine amino acid substitution at an amino acidcorresponding to amino acid residue Val126 of SEQ ID NO: 1; vii. anaspartic acid to glycine amino acid substitution at an amino acidcorresponding to amino acid residue Asp134 of SEQ ID NO: 1; and viii. alysine to asparagine amino acid substitution at an amino acidcorresponding to amino acid residue Lys186 of SEQ ID NO: 1. In certainembodiments that may be combined with any of the preceding embodiments,the individual has a heterozygous variant of TREM2, wherein the variantcomprises a guanine nucleotide deletion at a nucleotide corresponding tonucleotide residue G313 of the nucleic acid sequence encoding SEQ ID NO:1; a guanine nucleotide deletion at a nucleotide corresponding tonucleotide residue G267 of the nucleic acid sequence encoding SEQ ID NO:1; or both. In certain embodiments that may be combined with any of thepreceding embodiments, the individual has a heterozygous variant ofDAP12, wherein the variant comprises one or more variants selected fromthe group consisting of: i. a methionine to threonine substitution at anamino acid corresponding to amino acid residue Met1 of SEQ ID NO: 2; ii.a glycine to arginine amino acid substitution at an amino acidcorresponding to amino acid residue Gly49 of SEQ ID NO: 2; iii. adeletion within exons 1-4 of the nucleic acid sequence encoding SEQ IDNO: 2; iv. an insertion of 14 amino acid residues at exon 3 of thenucleic acid sequence encoding SEQ ID NO: 2; and v. a guanine nucleotidedeletion at a nucleotide corresponding to nucleotide residue G141 of thenucleic acid sequence encoding SEQ ID NO: 2.

In certain embodiments that may be combined with any of the precedingembodiments, the cancer is selected from the group consisting of bladdercancer, brain cancer, breast cancer, colon cancer, rectal cancer,endometrial cancer, kidney cancer, renal cell cancer, renal pelviscancer, leukemia, lung cancer, melanoma, non-Hodgkin's lymphoma,pancreatic cancer, prostate cancer, ovarian cancer, fibrosarcoma, andthyroid cancer. In certain embodiments that may be combined with any ofthe preceding embodiments, the method further comprises administering tothe individual at least one antibody that specifically binds to aninhibitory checkpoint molecule, and/or another standard orinvestigational anti-cancer therapy. In certain embodiments that may becombined with any of the preceding embodiments, the at least oneantibody that specifically binds to an inhibitory checkpoint molecule isadministered in combination with the isolated antibody. In certainembodiments that may be combined with any of the preceding embodiments,the at least one antibody that specifically binds to an inhibitorycheckpoint molecule is selected from the group consisting of ananti-PD-L1 antibody, an anti-CTLA4 antibody, an anti-PD-L2 antibody, ananti-PD-1 antibody, an anti-B7-H3 antibody, an anti-B7-H4 antibody, andanti-HVEM antibody, an anti-B- and T-lymphocyte attenuator (BTLA)antibody, an anti-Killer inhibitory receptor (KIR) antibody, ananti-GAL9 antibody, an anti-TIM3 antibody, an anti-A2AR antibody, ananti-LAG-3 antibody, an anti-phosphatidylserine antibody, an anti-CD27antibody, and any combination thereof. In certain embodiments that maybe combined with any of the preceding embodiments, the standard orinvestigational anti-cancer therapy is one or more therapies selectedfrom the group consisting of radiotherapy, cytotoxic chemotherapy,targeted therapy, imatinib (Gleevec®), trastuzumab (Herceptin®),adoptive cell transfer (ACT), chimeric antigen receptor T cell transfer(CAR-T), vaccine therapy, and cytokine therapy. In certain embodimentsthat may be combined with any of the preceding embodiments, the methodfurther comprises administering to the individual at least one antibodythat specifically binds to an inhibitory cytokine. In certainembodiments that may be combined with any of the preceding embodiments,the at least one antibody that specifically binds to an inhibitorycytokine is administered in combination with the isolated antibody. Incertain embodiments that may be combined with any of the precedingembodiments, the at least one antibody that specifically binds to aninhibitory cytokine is selected from the group consisting of ananti-CCL2 antibody, an anti-CSF-1 antibody, an anti-IL-2 antibody, andany combination thereof. In certain embodiments that may be combinedwith any of the preceding embodiments, the method further comprisesadministering to the individual at least one agonistic antibody thatspecifically binds to a stimulatory checkpoint protein. In certainembodiments that may be combined with any of the preceding embodiments,the at least one agonistic antibody that specifically binds to astimulatory checkpoint protein is administered in combination with theisolated antibody. In certain embodiments that may be combined with anyof the preceding embodiments, the at least one agonistic antibody thatspecifically binds to a stimulatory checkpoint protein is selected fromthe group consisting of an agonist anti-CD40 antibody, an agonistanti-OX40 antibody, an agonist anti-ICOS antibody, an agonist anti-CD28antibody, an agonist anti-CD137/4-1BB antibody, an agonist anti-CD27antibody, an agonist anti-glucocorticoid-induced TNFR-related proteinGITR antibody, and any combination thereof. In certain embodiments thatmay be combined with any of the preceding embodiments, the methodfurther comprises administering to the individual at least onestimulatory cytokine. In certain embodiments that may be combined withany of the preceding embodiments, the at least one stimulatory cytokineis administered in combination with the isolated antibody. In certainembodiments that may be combined with any of the preceding embodiments,the at least one stimulatory cytokine is selected from the groupconsisting of TNF-α, IL-10, IL-6, IL-8, CRP, TGF-beta members of thechemokine protein families, IL20 family member, IL-33, LIF, OSM, CNTF,TGF-beta, IL-11, IL-12, IL-17, IL-8, CRP, IFN-α, IFN-β, IL-2, IL-18,GM-CSF, G-CSF, and any combination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows an amino acid sequence alignment between the human TREM2protein (SEQ ID NO: 426) and the human NCTR2 protein (SEQ ID NO: 427),depicting the homology between the two proteins. The consensus sequenceis SEQ ID NO: 446. FIG. 1B shows a structure-based sequence alignmentbetween several TREM proteins and other members of the IgV family,TREM-1_human (SEQ ID NO: 429), TREM-2_human (SEQ ID NO: 430),TREM-1_mouse (SEQ ID NO: 431), TREM-2_mouse (SEQ ID NO: 432),TREM-3_mouse (SEQ ID NO: 433), NKp44 (SEQ ID NO: 434), aTCR_human (SEQID NO: 435), bTCR_human (SEQ ID NO: 436), gTCR_human (SEQ ID NO: 437),dTCR_human (SEQ ID NO: 438), Vd_human (SEQ ID NO: 439), hIGG1_mouse (SEQID NO: 440), lIGG1_mouse (SEQ ID NO: 441), CD8_human (SEQ ID NO: 442),and CTLA4_human (SEQ ID NO: 443). The amino acid residue numbering isconsistent with the mature sequence of the human TREM1 protein. Thesecondary structure elements of TREM1 are illustrated as arrows for theβ strands and cylinders for a helices. Amino acid residues involved inhomo- and heterodimer formation are shown on black background. Cysteineresidues that form disulfide bonds and that are conserved for the V-typeIg fold, are depicted in bold and marked with asterisks. Gaps areindicated by “-”. M-1 residues violating antibody-like dimer formationmode are marked with closed triangles as (e.g., Radaev et al., (2003)Structure. 11(12):1527-1535).

FIG. 2A shows an amino acid sequence alignment between the human TREM1protein (SEQ ID NO: 428) and the human TREM2 protein (SEQ ID NO: 426),depicting the homology between the two proteins. The consensus sequenceis SEQ ID NO: 447. FIG. 2B shows the amino acid sequences of the heavychain variable regions of antibodies Ab21 and Ab52. The CDR sequencesare underlined in each sequence. Sequence regions in-between eachunderlined CDR sequence correspond to the framework regions. FIG. 2Cshows the amino acid sequences of the light chain variable regions ofantibodies Ab21 and Ab52. The CDR sequences are underlined in eachsequence. Sequence regions in-between each underlined CDR sequencecorrespond to the framework regions.

FIG. 3A shows FACS histograms demonstrating binding of TREM2 antibodiesAb21, Ab52, Ab16, Ab20, Ab66, and Ab68 to a mouse cell line (BWZ T2)expressing recombinant mouse TREM2. FIG. 3B shows antibodies Ab21 andAb52 binding to WT (Trem+/+) and TREM2 deficient (TREM2−/−) bone marrowderived mouse macrophages (BMMac). Shaded histograms represent the TREM2antibody negative population. Black outlined histograms represent theTREM2 antibody positive population.

FIG. 4A shows FACS histograms demonstrating binding of TREM2 antibodiesAb21, Ab52, Ab43, and Ab60 to a human cell line (293) expressingrecombinant human TREM2-DAP12 fusion protein. Shaded histogramsrepresent a TREM2 antibody negative population. Black outlinedhistograms represent a TREM2 antibody positive population. FIG. 4B showsantibodies Ab21, Ab52, Ab43, and Ab60 binding to primary human dendriticcells (hDCs). Shaded histograms represent secondary antibody alonenegative control. Black outlined histograms represent the TREM2 antibodypositive population.

FIG. 5A shows FACS dot plots demonstrating expression of cell surfacemarkers CD83 and CD86 on human dendritic cells (DCs) after incubationwith plate-bound TREM2 antibodies Ab21 or Ab52. Antibody Ab88 representsthe negative isotype control. Plots were gated on CD11c⁺ HLA-DR⁺ LIN⁻DCs. Percentage of cells within the CD83+CD86+ gate is displayed on eachplot. FIG. 5B shows FACS histograms demonstrating expressing of cellsurface marker CD86 on human dendritic cells (DCs) after incubation withcross-linked TREM2 antibodies Ab21 or Ab52. Antibodies were cross-linkedwith anti-human secondary antibody. Antibody Ab88 represents thenegative isotype control.

FIG. 6A shows Syk phosphorylation as determine by western blot inwild-type and TREM2 deficient (Trem2−/−) mouse (left and center panels)and human (right panel) macrophages after incubation with TREM2antibodies Ab21 and Ab52. Antibodies Ab89 and Ab92 are non-agonisticnegative controls. FIG. 6B shows Syk phosphorylation as determine bywestern blot in primary human dendritic cells after incubation withTREM2 antibodies Ab21 and Ab52.

FIG. 7A shows DAP12 phosphorylation as determine by western blot inmouse macrophages after incubation with TREM2 antibody Ab52. FIG. 7Bshows DAP12 phosphorylation as determine by western blot in wild-typeand TREM2 deficient (Trem2−/−) mouse macrophages after incubation withTREM2 antibody Ab21.

FIG. 8 shows competitive binding between TREM2 antibodies and E. colibacteria expressing putative TREM2 ligand with mouse and human celllines expressing TREM2. Bacterial binding is expressed as a percentageof control. Average of two independent experiments; black bars: nodifference to isotype control, red bars: significantly different fromisotype controls (ANOVA).

FIG. 9A shows protein levels of inflammatory cytokines TNFa, IL-6,IL-10, and MCP-1 secreted in response to stimulation of WT and TREM2 KOmacrophages with inflammatory mediators LPS or Zymosan. FIG. 9B showsprotein levels of inflammatory cytokines IL-6 and TNFa secreted inresponse to stimulation of WT, TREM2 heterozygous (Het), and TREM2 KOmacrophages with the cytokines IL-4 or IFNg.

FIG. 10A shows FACS data demonstrating expression of cell surfacemarkers CD86 and CD206 on WT, TREM2 heterozygous (Het), and TREM2 KOmacrophages after stimulation with the cytokines IL-4 or IFNg. FIG. 10Bshows expression of cell surface marker CD86 on WT and TREM2 KOmacrophages after stimulation with the inflammatory mediators LPS orZymosan.

FIG. 11A shows numbers of live WT, TREM2 heterozygous (TREM2+/−), andTREM2 KO (TREM2−/−) macrophages after culture in growth factor MCSF forthe indicated number of days. FIG. 11B shows FACS plots demonstratingstaining of WT, TREM2 heterozygous (TREM2+/−), and TREM2 KO (TREM2−/−)macrophages after culture in MCSF for 6 days (+MCSF) or in MCSF for 4days, followed by no MCSF for 36 hours (−MCSF). Percentage of livemacrophages within the CD11b+DAPI− gate is indicated on each plot. FIG.11C shows luminescence levels detected in a luciferase viability assayafter culture of WT and TREM2 KO dendritic cells, M macrophages, and M2macrophages in growth factors GM-CSF, M-CSF, or M-CSF+IL-4,respectively. FIG. 11D shows the frequency of live WT, TREM2heterozygous (Het), and TREM2 KO macrophages (CD11b+) after culture ininflammatory mediators IFNg, LPS, or Zymosan.

FIG. 12 shows phagocytosis of apoptotic cells and E. coli by wild-type(WT) and TREM2 KO (TREM2−/−) bone marrow derived macrophages (BMmacs)cultured without MCSF.

FIG. 13 shows an epitope map of TREM2 antibody Ab52.

FIG. 14 shows Syk phosphorylation as determined by Western blot inwild-type and TREM2 deficient (Trem2−/−) mouse macrophages afterincubation with TREM2 antibodies MAB17291 (RD) or 78.18, demonstratingthat antibody 78.18 does not induce Syk phosphorylation or TREM2signaling.

FIG. 15A shows Fortebio analysis demonstrating simultaneous binding ofantibody MAB17291 and antibody Ab21 to TREM2-Fc. FIG. 15A shows Fortebioanalysis demonstrating simultaneous binding of antibody MAB17291 andantibody Ab52 to TREM2-Fc.

FIG. 16 shows the percent increased survival of wild-type (WT) and TREM2knock-out (KO) mouse bone marrow derived macrophages cultured in thepresence of plate bound, cross-linked TREM2 antibody Ab21 or Ab52 Fabsand M-CSF. Antibody Ab88 represents the negative isotype control.

FIG. 17A shows the luminescence viability assay of mouse bone marrowderived macrophages cultured in the presence of soluble,non-cross-linked TREM2 antibody Ab21 or Ab52 Fabs and M-CSF. AntibodyAb99 represents the negative isotype control. FIG. 17B shows theluminescence viability assay of mouse bone marrow derived macrophagescultured in the presence of soluble, full-length TREM2 antibody Ab21 orAb52 and M-CSF. Antibody Ab91 represents the negative isotype control.The “NT” dotted line indicates the average viability obtained withuntreated macrophages (no antibody added). The “no MCSF” dotted lineindicates the average viability obtained when macrophages are culturedin the absence of M-CSF.

FIG. 18A shows induction of TREM2-dependent gene expression by platebound, full-length anti-TREM2 antibodies Ab21 and Ab52 using aluciferase reporter gene in a cell-based assay. FIG. 18B shows inductionof TREM2-dependent gene expression by plate bound phosphatidylserine(PS). FIG. 18C shows activation of TREM2-dependent gene IL-6 in mousemacrophages by plate bound, Fab anti-TREM2 antibodies Ab21 and Ab52Fabs. FIG. 18D shows activation of TREM2-dependent gene MCP-1 in mousemacrophages by plate bound, Fab anti-TREM2 antibodies Ab21 and Ab52Fabs. Data in FIGS. 18C and 18D are shown as means±SD; n=3 mice pergroup. In FIGS. 18C and 18D “no Ab” indicates negative control with noantibody treatment, “21” indicates treatment with Ab21 Fab, “52”indicates treatment with Ab52 Fab, and “ctr” indicates treatment withcontrol antibody Fab.

FIG. 19 shows inhibition of TREM2-dependent gene expression by soluble,full-length anti-TREM2 antibodies Ab21 and Ab52 using a luciferasereporter gene in a cell-based assay.

FIG. 20A shows the amino acid sequences of the heavy chain variableregions of TREM2 antibodies. The CDR sequences are underlined in eachsequence. Sequence regions in-between each underlined CDR sequencecorrespond to the framework regions. FIG. 20B shows the amino acidsequences of the light chain variable regions of TREM2 antibodies. TheCDR sequences are underlined in each sequence. Sequence regionsin-between each underlined CDR sequence correspond to the frameworkregions.

FIG. 21A shows FACS histograms demonstrating binding of TREM2 antibodiesAb1, Ab9, Ab14, Ab22, Ab45, and Ab65 to a mouse cell line (BWZ T2)expressing recombinant mouse TREM2. FIG. 21B shows antibodies Ab1, Ab9,Ab14, Ab22, Ab45, and Ab65 binding to WT (Trem+/+) and TREM2 deficient(TREM2−/−) bone marrow derived mouse macrophages (BMMac). Antibody Ab88represents the negative isotype control. Shaded histograms represent theTREM2 antibody negative population. Black outlined histograms representthe TREM2 antibody positive population.

FIG. 22A shows FACS histograms demonstrating binding of TREM2 antibodiesAb1, Ab9, Ab14, Ab22, Ab43, Ab45, Ab60, and Ab65 to a human cell line(293) expressing recombinant human TREM2-DAP12 fusion protein. Shadedhistograms represent a TREM2 antibody negative population. Blackoutlined histograms represent a TREM2 antibody positive population. FIG.22B shows antibodies Ab1, Ab9, Ab14, Ab22, Ab43, Ab45, Ab60, and Ab65binding to primary human dendritic cells (hDCs). Antibody Ab88represents the negative isotype control. Shaded histograms representsecondary antibody alone negative control. Black outlined histogramsrepresent the TREM2 antibody positive population.

FIG. 23A shows FACS dot plots demonstrating expression of cell surfacemarkers CD83 and CD86 on human dendritic cells (DCs) after incubationwith plate-bound TREM2 antibodies Ab1, Ab9, Ab14, Ab22, Ab45, and Ab65.Antibody Ab88 represents the negative isotype control. Plots were gatedon CD11c⁺ HLA-DR⁺ LIN⁻ DCs. Percentage of cells within the CD83+CD86+gate is displayed on each plot. FIG. 23B shows FACS histogramsdemonstrating expression of cell surface marker CD86 on human dendriticcells (DCs) after incubation with cross-linked TREM2 antibodies Ab1,Ab9, Ab14, Ab22, Ab45, and Ab65. Antibodies were cross-linked withanti-human secondary antibody. Antibody Ab88 represents the negativeisotype control.

FIG. 24A shows Syk phosphorylation as determined by western blot inwild-type and TREM2 deficient (Trem2−/−) mouse macrophages afterincubation with TREM2 antibodies Ab1, Ab9, or Ab45. Antibodies Ab89 andAb92 are negative isotype controls. FIG. 24B shows Syk phosphorylationas determined by western blot in human macrophages after incubation withTREM2 antibodies Ab1, Ab9, Ab14, Ab20, Ab22, Ab45, and Ab65. AntibodiesAb16 and Ab77 are non-agonistic negative controls. FIG. 24C shows Sykphosphorylation as determined by western blot in primary human dendriticcells after incubation with TREM2 antibodies Ab1, Ab5, Ab9, Ab22, Ab45,or Ab65.

FIG. 25 shows competitive binding between TREM2 antibodies Ab1, Ab9,Ab14, Ab22, Ab45, Ab65, Ab66, and Ab68 and E. coli cells expressingputative TREM2 ligand to mouse and human cell lines expressing TREM2.Bacterial binding is expressed as a percentage of control. Average oftwo independent experiments; black bars: no difference to isotypecontrol, red bars: significantly different from isotype controls(ANOVA).

FIG. 26A shows DAP12 phosphorylation as determined by western blot inmouse macrophages after incubation with TREM2 antibody Ab45 or Ab65.FIG. 26B shows DAP12 phosphorylation as determine by western blot inwild-type and TREM2 deficient (Trem2−/−) mouse macrophages afterincubation with TREM2 antibody Ab1, Ab9, Ab22, or Ab45.

FIG. 27A shows an epitope map of TREM2 antibodies Ab1 and Ab9. FIG. 27Bshows an epitope map of TREM2 antibodies Ab45 and Ab65.

FIG. 28 shows Fortebio analysis demonstrating simultaneous binding ofantibody MAB17291 and antibodies Ab1, Ab9, Ab14, Ab22, Ab45, and Ab65 toTREM2-Fc. Control antibodies Ab63 and Ab87 did not simultaneously bindto TREM2-Fc.

FIG. 29 shows the percent increased survival of wild-type (WT) and TREM2knock-out (KO) mouse bone marrow derived macrophages cultured in thepresence of plate bound, cross-linked TREM2 antibody Ab22, Ab45, or Ab65Fabs and M-CSF. Antibody Ab88 represents the negative isotype control.

FIG. 30A shows the luminescence viability assay of mouse bone marrowderived macrophages cultured in the presence of soluble,non-cross-linked TREM2 antibody Fabs and M-CSF. Antibody Ab99 representsthe negative isotype control. FIG. 30B shows the luminescence viabilityassay of mouse bone marrow derived macrophages cultured in the presenceof soluble, full-length TREM2 antibodies and M-CSF. Antibody Ab91represents the negative isotype control. The “NT” dotted line indicatesthe average viability obtained with untreated macrophages (no antibodyadded). The “no MCSF” dotted line indicates the average viabilityobtained when macrophages are cultured in the absence of M-CSF.

FIG. 31A shows induction of TREM2-dependent gene expression by platebound, full-length anti-TREM2 antibodies using a luciferase reportergene in a cell-based assay. FIG. 31B shows induction of TREM2-dependentgene expression by plate bound, full-length anti-TREM2 antibodies usinga luciferase reporter gene in a cell-based assay. FIG. 31C showsinduction of TREM2-dependent gene expression by plate boundphosphatidylserine (PS).

FIG. 32 shows inhibition of TREM2-dependent gene expression by soluble,full-length anti-TREM2 antibodies using a luciferase reporter gene in acell-based assay.

FIG. 33 shows competitive interactions between TREM2 antibodies Ab22 andAb45 and Phosphatidylserine (PS) or Sphingomyelin (SM) in mouse andhuman cell lines expressing TREM2.

FIG. 34A shows activation of TREM2-dependent gene IL-6 in mousemacrophages by plate bound, Fab anti-TREM2 antibodies Ab22 and Ab65Fabs. FIG. 34B shows activation of TREM2-dependent gene MCP-1 in mousemacrophages by plate bound, Fab anti-TREM2 antibodies Ab22 and Ab65Fabs. Data in FIGS. 34A and 34B are shown as means±SD; n=3 mice pergroup. In FIGS. 34A and 34B “no Ab” indicates negative control with noantibody treatment, “22” indicates treatment with Ab22 Fab, “65”indicates treatment with Ab65 Fab, and “ctr” indicates treatment withcontrol antibody Fab.

DETAILED DESCRIPTION OF THE INVENTION General Techniques

The techniques and procedures described or referenced herein aregenerally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, thewidely utilized methodologies described in Sambrook et al., MolecularCloning: A Laboratory Manual 3d edition (2001) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.; Current Protocols inMolecular Biology (F. M. Ausubel, et al. eds., (2003)); the seriesMethods in Enzymology (Academic Press, Inc.): PCR 2: A PracticalApproach (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)),Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and AnimalCell Culture (R. I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; CellBiology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press;Animal Cell Culture (R. I. Freshney), ed., 1987); Introduction to Celland Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press;Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B.Griffiths, and D. G. Newell, eds., 1993-8) J. Wiley and Sons; Handbookof Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); GeneTransfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos,eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds.,1994); Current Protocols in Immunology (J. E. Coligan et al., eds.,1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999);Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P.Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRLPress, 1988-1989); Monoclonal Antibodies: A Practical Approach (P.Shepherd and C. Dean, eds., Oxford University Press, 2000); UsingAntibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold SpringHarbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D.Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principlesand Practice of Oncology (V. T. DeVita et al., eds., J. B. LippincottCompany, 1993).

Definitions

As used herein, the term “preventing” includes providing prophylaxiswith respect to occurrence or recurrence of a particular disease,disorder, or condition in an individual. An individual may bepredisposed to, susceptible to a particular disease, disorder, orcondition, or at risk of developing such a disease, disorder, orcondition, but has not yet been diagnosed with the disease, disorder, orcondition.

As used herein, an individual “at risk” of developing a particulardisease, disorder, or condition may or may not have detectable diseaseor symptoms of disease, and may or may not have displayed detectabledisease or symptoms of disease prior to the treatment methods describedherein. “At risk” denotes that an individual has one or more riskfactors, which are measurable parameters that correlate with developmentof a particular disease, disorder, or condition, as known in the art. Anindividual having one or more of these risk factors has a higherprobability of developing a particular disease, disorder, or conditionthan an individual without one or more of these risk factors.

As used herein, the term “treatment” refers to clinical interventiondesigned to alter the natural course of the individual being treatedduring the course of clinical pathology. Desirable effects of treatmentinclude decreasing the rate of progression, ameliorating or palliatingthe pathological state, and remission or improved prognosis of aparticular disease, disorder, or condition. An individual issuccessfully “treated”, for example, if one or more symptoms associatedwith a particular disease, disorder, or condition are mitigated oreliminated.

An “effective amount” refers to at least an amount effective, at dosagesand for periods of time necessary, to achieve the desired therapeutic orprophylactic result. An effective amount can be provided in one or moreadministrations.

A “therapeutically effective amount” is at least the minimumconcentration required to effect a measurable improvement of aparticular disease, disorder, or condition. A therapeutically effectiveamount herein may vary according to factors such as the disease state,age, sex, and weight of the patient, and the ability of the anti-TREM2and/or anti-DAP12 antibody to elicit a desired response in theindividual. A therapeutically effective amount is also one in which anytoxic or detrimental effects of the anti-TREM2 and/or anti-DAP12antibody are outweighed by the therapeutically beneficial effects.

As used herein, administration “in conjunction” with another compound orcomposition includes simultaneous administration and/or administrationat different times. Administration in conjunction also encompassesadministration as a co-formulation or administration as separatecompositions, including at different dosing frequencies or intervals,and using the same route of administration or different routes ofadministration.

An “individual” for purposes of treatment, prevention, or reduction ofrisk refers to any animal classified as a mammal, including humans,domestic and farm animals, and zoo, sport, or pet animals, such as dogs,horses, rabbits, cattle, pigs, hamsters, gerbils, mice, ferrets, rats,cats, and the like. Preferably, the individual is human.

The term “immunoglobulin” (Ig) is used interchangeably with “antibody”herein. The term “antibody” herein is used in the broadest sense andspecifically covers monoclonal antibodies, polyclonal antibodies,multispecific antibodies (e.g., bispecific antibodies) formed from atleast two intact antibodies, and antibody fragments so long as theyexhibit the desired biological activity.

The basic 4-chain antibody unit is a heterotetrameric glycoproteincomposed of two identical light (L) chains and two identical heavy (H)chains. The pairing of a V_(H) and V_(L) together forms a singleantigen-binding site. For the structure and properties of the differentclasses of antibodies, see, e.g., Basic and Clinical Immunology, 8thEd., Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.),Appleton & Lange, Norwalk, Conn., 1994, page 71 and Chapter 6.

The L chain from any vertebrate species can be assigned to one of twoclearly distinct types, called kappa (“κ”) and lambda (“λ”), based onthe amino acid sequences of their constant domains. Depending on theamino acid sequence of the constant domain of their heavy chains (CH),immunoglobulins can be assigned to different classes or isotypes. Thereare five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, havingheavy chains designated alpha (“α”), delta (“δ”), epsilon (“ε”), gamma(“γ”) and mu (“μ”), respectively. The γ and α classes are furtherdivided into subclasses (isotypes) on the basis of relatively minordifferences in the CH sequence and function, e.g., humans express thefollowing subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Thesubunit structures and three dimensional configurations of differentclasses of immunoglobulins are well known and described generally in,for example, Abbas et al., Cellular and Molecular Immunology, 4^(th) ed.(W.B. Saunders Co., 2000).

“Native antibodies” are usually heterotetrameric glycoproteins of about150,000 daltons, composed of two identical light (L) chains and twoidentical heavy (H) chains. Each light chain is linked to a heavy chainby one covalent disulfide bond, while the number of disulfide linkagesvaries among the heavy chains of different immunoglobulin isotypes. Eachheavy and light chain also has regularly spaced intrachain disulfidebridges. Each heavy chain has at one end a variable domain (V_(H))followed by a number of constant domains. Each light chain has avariable domain at one end (V_(L)) and a constant domain at its otherend; the constant domain of the light chain is aligned with the firstconstant domain of the heavy chain, and the light chain variable domainis aligned with the variable domain of the heavy chain. Particular aminoacid residues are believed to form an interface between the light chainand heavy chain variable domains.

An “isolated” antibody, such as an isolated anti-TREM2 and/or anti-DAP12antibody of the present disclosure, is one that has been identified,separated and/or recovered from a component of its productionenvironment (e.g., naturally or recombinantly). Preferably, the isolatedpolypeptide is free of association with all other contaminant componentsfrom its production environment. Contaminant components from itsproduction environment, such as those resulting from recombinanttransfected cells, are materials that would typically interfere withresearch, diagnostic or therapeutic uses for the antibody, and mayinclude enzymes, hormones, and other proteinaceous or non-proteinaceoussolutes. In preferred embodiments, the polypeptide will be purified: (1)to greater than 95% by weight of antibody as determined by, for example,the Lowry method, and in some embodiments, to greater than 99% byweight; (2) to a degree sufficient to obtain at least 15 residues ofN-terminal or internal amino acid sequence by use of a spinning cupsequenator, or (3) to homogeneity by SDS-PAGE under non-reducing orreducing conditions using Coomassie blue or, preferably, silver stain.Isolated antibody includes the antibody in situ within recombinantT-cells since at least one component of the antibody's naturalenvironment will not be present. Ordinarily, however, an isolatedpolypeptide or antibody will be prepared by at least one purificationstep.

The “variable region” or “variable domain” of an antibody, such as ananti-TREM2 and/or anti-DAP12 antibody of the present disclosure, refersto the amino-terminal domains of the heavy or light chain of theantibody. The variable domains of the heavy chain and light chain may bereferred to as “V_(H)” and “V_(L)”, respectively. These domains aregenerally the most variable parts of the antibody (relative to otherantibodies of the same class) and contain the antigen binding sites.

The term “variable” refers to the fact that certain segments of thevariable domains differ extensively in sequence among antibodies, suchas anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure.The V domain mediates antigen binding and defines the specificity of aparticular antibody for its particular antigen. However, the variabilityis not evenly distributed across the entire span of the variabledomains. Instead, it is concentrated in three segments calledhypervariable regions (HVRs) both in the light-chain and the heavy chainvariable domains. The more highly conserved portions of variable domainsare called the framework regions (FR). The variable domains of nativeheavy and light chains each comprise four FR regions, largely adopting abeta-sheet configuration, connected by three HVRs, which form loopsconnecting, and in some cases forming part of, the beta-sheet structure.The HVRs in each chain are held together in close proximity by the FRregions and, with the HVRs from the other chain, contribute to theformation of the antigen-binding site of antibodies (see Kabat et al.,Sequences of Immunological Interest, Fifth Edition, National Instituteof Health, Bethesda, Md. (1991)). The constant domains are not involveddirectly in the binding of antibody to an antigen, but exhibit variouseffector functions, such as participation of the antibody inantibody-dependent-cellular toxicity.

The term “monoclonal antibody” as used herein refers to an antibody,such as a monoclonal anti-TREM2 and/or anti-DAP12 antibody of thepresent disclosure, obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally occurringmutations and/or post-translation modifications (e.g., isomerizations,amidations, etc.) that may be present in minor amounts. Monoclonalantibodies are highly specific, being directed against a singleantigenic site. In contrast to polyclonal antibody preparations whichtypically include different antibodies directed against differentdeterminants (epitopes), each monoclonal antibody is directed against asingle determinant on the antigen. In addition to their specificity, themonoclonal antibodies are advantageous in that they are synthesized bythe hybridoma culture, uncontaminated by other immunoglobulins. Themodifier “monoclonal” indicates the character of the antibody as beingobtained from a substantially homogeneous population of antibodies, andis not to be construed as requiring production of the antibody by anyparticular method. For example, the monoclonal antibodies to be used inaccordance with the present invention may be made by a variety oftechniques, including, for example, the hybridoma method (e.g., Kohlerand Milstein., Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14(3):253-260 (1995), Harlow et al., Antibodies: A Laboratory Manual,(Cold Spring Harbor Laboratory Press, 2d ed. 1988); Hammerling et al.,in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y.,1981)), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567),phage-display technologies (see, e.g., Clackson et al., Nature,352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992);Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol.Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Nat'l Acad. Sci. USA101(34): 12467-472 (2004); and Lee et al., J. Immunol. Methods284(1-2):119-132 (2004), and technologies for producing human orhuman-like antibodies in animals that have parts or all of the humanimmunoglobulin loci or genes encoding human immunoglobulin sequences(see, e.g., WO 1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741;Jakobovits et al., Proc. Nat'l Acad. Sci. USA 90:2551 (1993); Jakobovitset al., Nature 362:255-258 (1993); Bruggemann et al., Year in Immunol.7:33 (1993); U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126;5,633,425; and U.S. Pat. No. 5,661,016; Marks et al., Bio/Technology10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison,Nature 368:812-813 (1994); Fishwild et al., Nature Biotechnol.14:845-851 (1996); Neuberger, Nature Biotechnol. 14:826 (1996); andLonberg and Huszar, Intern. Rev. Immunol. 13:65-93 (1995).

The terms “full-length antibody,” “intact antibody” or “whole antibody”are used interchangeably to refer to an antibody, such as an anti-TREM2and/or anti-DAP12 antibody of the present disclosure, in itssubstantially intact form, as opposed to an antibody fragment.Specifically whole antibodies include those with heavy and light chainsincluding an Fc region. The constant domains may be native sequenceconstant domains (e.g., human native sequence constant domains) or aminoacid sequence variants thereof. In some cases, the intact antibody mayhave one or more effector functions.

An “antibody fragment” comprises a portion of an intact antibody,preferably the antigen binding and/or the variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂ andFv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870,Example 2; Zapata et al., Protein Eng. 8(10):1057-1062 (1995));single-chain antibody molecules and multispecific antibodies formed fromantibody fragments.

Papain digestion of antibodies, such as anti-TREM2 and/or anti-DAP12antibodies of the present disclosure, produces two identicalantigen-binding fragments, called “Fab” fragments, and a residual “Fc”fragment, a designation reflecting the ability to crystallize readily.The Fab fragment consists of an entire L chain along with the variableregion domain of the H chain (V_(H)), and the first constant domain ofone heavy chain (C_(H)1). Each Fab fragment is monovalent with respectto antigen binding, i.e., it has a single antigen-binding site. Pepsintreatment of an antibody yields a single large F(ab′)₂ fragment whichroughly corresponds to two disulfide linked Fab fragments havingdifferent antigen-binding activity and is still capable of cross-linkingantigen. Fab′ fragments differ from Fab fragments by having a fewadditional residues at the carboxy terminus of the C_(H)1 domainincluding one or more cysteines from the antibody hinge region. Fab′-SHis the designation herein for Fab′ in which the cysteine residue(s) ofthe constant domains bear a free thiol group. F(ab′)₂ antibody fragmentsoriginally were produced as pairs of Fab′ fragments which have hingecysteines between them. Other chemical couplings of antibody fragmentsare also known.

The Fc fragment comprises the carboxy-terminal portions of both H chainsheld together by disulfides. The effector functions of antibodies aredetermined by sequences in the Fc region, the region which is alsorecognized by Fc receptors (FcR) found on certain types of cells.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This fragment consists of a dimerof one heavy- and one light-chain variable region domain in tight,non-covalent association. From the folding of these two domains emanatesix hypervariable loops (3 loops each from the H and L chain) thatcontribute the amino acid residues for antigen binding and conferantigen binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three HVRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

“Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibodyfragments that comprise the VH and VL antibody domains connected into asingle polypeptide chain. Preferably, the sFv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains,which enables the sFv to form the desired structure for antigen binding.For a review of the sFv, see Plückthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994).

“Functional fragments” of antibodies, such as anti-TREM2 and/oranti-DAP12 antibodies of the present disclosure, comprise a portion ofan intact antibody, generally including the antigen binding or variableregion of the intact antibody or the F region of an antibody whichretains or has modified FcR binding capability. Examples of antibodyfragments include linear antibody, single-chain antibody molecules andmultispecific antibodies formed from antibody fragments.

The term “diabodies” refers to small antibody fragments prepared byconstructing sFv fragments (see preceding paragraph) with short linkers(about 5-10) residues) between the V_(H) and V_(L) domains such thatinter-chain but not intra-chain pairing of the V domains is achieved,thereby resulting in a bivalent fragment, i.e., a fragment having twoantigen-binding sites. Bispecific diabodies are heterodimers of two“crossover” sFv fragments in which the V_(H) and V_(L) domains of thetwo antibodies are present on different polypeptide chains. Diabodiesare described in greater detail in, for example, EP 404,097; WO93/11161; Hollinger et al., Proc. Nat'l Acad. Sci. USA 90:6444-48(1993).

As used herein, a “chimeric antibody” refers to an antibody(immunoglobulin), such as a chimeric anti-TREM2 and/or anti-DAP12antibody of the present disclosure, in which a portion of the heavyand/or light chain is identical with or homologous to correspondingsequences in antibodies derived from a particular species or belongingto a particular antibody class or subclass, while the remainder of thechain(s) is(are) identical with or homologous to corresponding sequencesin antibodies derived from another species or belonging to anotherantibody class or subclass, as well as fragments of such antibodies, solong as they exhibit the desired biological activity (U.S. Pat. No.4,816,567; Morrison et al., Proc. Nat'l Acad. Sci. USA, 81:6851-55(1984)). Chimeric antibodies of interest herein include PRIMATIZED®antibodies wherein the antigen-binding region of the antibody is derivedfrom an antibody produced by, e.g., immunizing macaque monkeys with anantigen of interest. As used herein, “humanized antibody” is used asubset of “chimeric antibodies.”

“Humanized” forms of non-human (e.g., murine) antibodies, such ashumanized forms of anti-TREM2 and/or anti-DAP12 antibodies of thepresent disclosure, are chimeric antibodies that contain minimalsequence derived from non-human immunoglobulin. In one embodiment, ahumanized antibody is a human immunoglobulin (recipient antibody) inwhich residues from an HVR of the recipient are replaced by residuesfrom an HVR of a non-human species (donor antibody) such as mouse, rat,rabbit or non-human primate having the desired specificity, affinity,and/or capacity. In some instances, FR residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues that are notfound in the recipient antibody or in the donor antibody. Thesemodifications may be made to further refine antibody performance, suchas binding affinity. In general, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin sequence, and all orsubstantially all of the FR regions are those of a human immunoglobulinsequence, although the FR regions may include one or more individual FRresidue substitutions that improve antibody performance, such as bindingaffinity, isomerization, immunogenicity, and the like. The number ofthese amino acid substitutions in the FR is typically no more than 6 inthe H chain, and in the L chain, no more than 3. The humanized antibodyoptionally will also comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin. Forfurther details, see, e.g., Jones et al., Nature 321:522-525 (1986);Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op.Struct. Biol. 2:593-596 (1992). See also, for example, Vaswani andHamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris,Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr.Op. Biotech. 5:428-433 (1994); and U.S. Pat. Nos. 6,982,321 and7,087,409.

A “human antibody” is one that possesses an amino-acid sequencecorresponding to that of an antibody, such as an anti-TREM2 and/oranti-DAP12 antibody of the present disclosure, produced by a humanand/or has been made using any of the techniques for making humanantibodies as disclosed herein. This definition of a human antibodyspecifically excludes a humanized antibody comprising non-humanantigen-binding residues. Human antibodies can be produced using varioustechniques known in the art, including phage-display libraries.Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J.Mol. Biol., 222:581 (1991). Also available for the preparation of humanmonoclonal antibodies are methods described in Cole et al., MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner etal., J. Immunol., 147(1):86-95 (1991). See also van Dijk and van deWinkel, Curr. Opin. Pharmacol. 5:368-74 (2001). Human antibodies can beprepared by administering the antigen to a transgenic animal that hasbeen modified to produce such antibodies in response to antigenicchallenge, but whose endogenous loci have been disabled, e.g., immunizedxenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 regardingXENOMOUSE™ technology). See also, for example, Li et al., Proc. Nat'lAcad. Sci. USA, 103:3557-3562 (2006) regarding human antibodiesgenerated via a human B-cell hybridoma technology.

The term “hypervariable region,” “HVR,” or “HV,” when used herein refersto the regions of an antibody-variable domain, such as that of ananti-TREM2 and/or anti-DAP12 antibody of the present disclosure, thatare hypervariable in sequence and/or form structurally defined loops.Generally, antibodies comprise six HVRs; three in the VH (H1, H2, H3),and three in the VL (L1, L2, L3). In native antibodies, H3 and L3display the most diversity of the six HVRs, and H3 in particular isbelieved to play a unique role in conferring fine specificity toantibodies. See, e.g., Xu et al., Immunity 13:37-45 (2000); Johnson andWu in Methods in Molecular Biology 248:1-25 (Lo, ed., Human Press,Totowa, N.J., 2003)). Indeed, naturally occurring camelid antibodiesconsisting of a heavy chain only are functional and stable in theabsence of light chain. See, e.g., Hamers-Casterman et al., Nature363:446-448 (1993) and Sheriff et al., Nature Struct. Biol. 3:733-736(1996).

A number of HVR delineations are in use and are encompassed herein. TheHVRs that are Kabat complementarity-determining regions (CDRs) are basedon sequence variability and are the most commonly used (Kabat et al.,supra). Chothia refers instead to the location of the structural loops(Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The AbM HVRsrepresent a compromise between the Kabat CDRs and Chothia structuralloops, and are used by Oxford Molecular's AbM antibody-modelingsoftware. The “contact” HVRs are based on an analysis of the availablecomplex crystal structures. The residues from each of these HVRs arenoted below.

Loop Kabat AbM Chothia Contact Ll L24-L34 L24-L34 L26-L32 L30-L36 L2L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1H31-H35B H26-H35B H26-H32 H30-H35B (Kabat numbering) H1 H31-H35 H26-H35H26-H32 H30-H35 (Chothia numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58H3 H95-H102 H95-H102 H96-H101 H93-H101

HVRs may comprise “extended HVRs” as follows: 24-36 or 24-34 (L1), 46-56or 50-56 (L2), and 89-97 or 89-96 (L3) in the VL, and 26-35 (H1), 50-65or 49-65 (a preferred embodiment) (H2), and 93-102, 94-102, or 95-102(H3) in the VH. The variable-domain residues are numbered according toKabat et al., supra, for each of these extended-HVR definitions.

“Framework” or “FR” residues are those variable-domain residues otherthan the HVR residues as herein defined.

The phrase “variable-domain residue-numbering as in Kabat” or“amino-acid-position numbering as in Kabat,” and variations thereof,refers to the numbering system used for heavy-chain variable domains orlight-chain variable domains of the compilation of antibodies in Kabatet al., supra. Using this numbering system, the actual linear amino acidsequence may contain fewer or additional amino acids corresponding to ashortening of, or insertion into, a FR or HVR of the variable domain.For example, a heavy-chain variable domain may include a single aminoacid insert (residue 52a according to Kabat) after residue 52 of H2 andinserted residues (e.g., residues 82a, 82b, and 82c, etc. according toKabat) after heavy-chain FR residue 82. The Kabat numbering of residuesmay be determined for a given antibody by alignment at regions ofhomology of the sequence of the antibody with a “standard” Kabatnumbered sequence.

The Kabat numbering system is generally used when referring to a residuein the variable domain (approximately residues 1-107 of the light chainand residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences ofImmunological Interest. 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991)). The “EU numbering system”or “EU index” is generally used when referring to a residue in animmunoglobulin heavy chain constant region (e.g., the EU index reportedin Kabat et al., supra). The “EU index as in Kabat” refers to theresidue numbering of the human IgG1 EU antibody. References to residuenumbers in the variable domain of antibodies means residue numbering bythe Kabat numbering system. References to residue numbers in theconstant domain of antibodies means residue numbering by the EUnumbering system (e.g., see United States Patent Publication No.2010-280227).

An “acceptor human framework” as used herein is a framework comprisingthe amino acid sequence of a VL or VH framework derived from a humanimmunoglobulin framework or a human consensus framework. An acceptorhuman framework “derived from” a human immunoglobulin framework or ahuman consensus framework may comprise the same amino acid sequencethereof, or it may contain pre-existing amino acid sequence changes. Insome embodiments, the number of pre-existing amino acid changes are 10or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 orless, 3 or less, or 2 or less. Where pre-existing amino acid changes arepresent in a VH, preferable those changes occur at only three, two, orone of positions 71H, 73H and 78H; for instance, the amino acid residuesat those positions may by 71A, 73T and/or 78A. In one embodiment, the VLacceptor human framework is identical in sequence to the VL humanimmunoglobulin framework sequence or human consensus framework sequence.

A “human consensus framework” is a framework that represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al., Sequences of Proteins of Immunological Interest, 5thEd. Public Health Service, National Institutes of Health, Bethesda, Md.(1991). Examples include for the VL, the subgroup may be subgroup kappaI, kappa II, kappa III or kappa IV as in Kabat et al., supra.Additionally, for the VH, the subgroup may be subgroup I, subgroup II,or subgroup III as in Kabat et al., supra.

An “amino-acid modification” at a specified position, e.g., of ananti-TREM2 and/or anti-DAP12 antibody of the present disclosure, refersto the substitution or deletion of the specified residue, or theinsertion of at least one amino acid residue adjacent the specifiedresidue. Insertion “adjacent” to a specified residue means insertionwithin one to two residues thereof. The insertion may be N-terminal orC-terminal to the specified residue. The preferred amino acidmodification herein is a substitution.

An “affinity-matured” antibody, such as an affinity matured anti-TREM2and/or anti-DAP12 antibody of the present disclosure, is one with one ormore alterations in one or more HVRs thereof that result in animprovement in the affinity of the antibody for antigen, compared to aparent antibody that does not possess those alteration(s). In oneembodiment, an affinity-matured antibody has nanomolar or even picomolaraffinities for the target antigen. Affinity-matured antibodies areproduced by procedures known in the art. For example, Marks et al.,Bio/Technology 10:779-783 (1992) describes affinity maturation by VH-and VL-domain shuffling. Random mutagenesis of HVR and/or frameworkresidues is described by, for example: Barbas et al. Proc Nat. Acad.Sci. USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155 (1995);Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al., J.Immunol. 154(7):3310-9 (1995); and Hawkins et al, J. Mol. Biol.226:889-896 (1992).

As use herein, the term “specifically recognizes” or “specificallybinds” refers to measurable and reproducible interactions such asattraction or binding between a target and an antibody, such as betweenan anti-TREM2 antibody and TREM2, or an anti-DAP12 antibody and DAP12that is determinative of the presence of the target in the presence of aheterogeneous population of molecules including biological molecules.For example, an antibody, such as an anti-TREM2 and/or anti-DAP12antibody of the present disclosure, that specifically or preferentiallybinds to a target or an epitope is an antibody that binds this target orepitope with greater affinity, avidity, more readily, and/or withgreater duration than it binds to other targets or other epitopes of thetarget. It is also understood by reading this definition that, forexample, an antibody (or a moiety) that specifically or preferentiallybinds to a first target may or may not specifically or preferentiallybind to a second target. As such, “specific binding” or “preferentialbinding” does not necessarily require (although it can include)exclusive binding. An antibody that specifically binds to a target mayhave an association constant of at least about 10³ M⁻¹ or 10⁴ M⁻¹,sometimes about 10⁵ M⁻¹ or 10⁶ M⁻¹, in other instances about 10⁶ M⁻¹ or10⁷ M⁻¹, about 10⁸ M⁻¹ to 10⁹ M⁻¹, or about 10¹⁰ M⁻¹ to 10¹¹ M⁻¹ orhigher. A variety of immunoassay formats can be used to selectantibodies specifically immunoreactive with a particular protein. Forexample, solid-phase ELISA immunoassays are routinely used to selectmonoclonal antibodies specifically immunoreactive with a protein. See,e.g., Harlow and Lane (1988) Antibodies, A Laboratory Manual, ColdSpring Harbor Publications, New York, for a description of immunoassayformats and conditions that can be used to determine specificimmunoreactivity.

As used herein, an “interaction” between a TREM2 protein, or DAP12protein, and a second protein encompasses, without limitation,protein-protein interaction, a physical interaction, a chemicalinteraction, binding, covalent binding, and ionic binding. As usedherein, an antibody “inhibits interaction” between two proteins when theantibody disrupts, reduces, or completely eliminates an interactionbetween the two proteins. An antibody of the present disclosure, orfragment thereof, “inhibits interaction” between two proteins when theantibody or fragment thereof binds to one of the two proteins.

An “agonist” antibody or an “activating” antibody is an antibody, suchas an agonist anti-TREM2 antibody or an agonist anti-DAP12 antibody ofthe present disclosure, that induces (e.g., increases) one or moreactivities or functions of the antigen after the antibody binds theantigen.

An “antagonist” antibody or a “blocking” antibody is an antibody, suchas an antagonist anti-TREM2 antibody or an antagonist anti-DAP12antibody of the present disclosure, that reduces or eliminates (e.g.,decreases) antigen binding to one or more ligand after the antibodybinds the antigen, and/or that reduces or eliminates (e.g., decreases)one or more activities or functions of the antigen after the antibodybinds the antigen.

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody, and vary with the antibodyisotype.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain, including native-sequence Fc regions andvariant Fc regions. Although the boundaries of the Fc region of animmunoglobulin heavy chain might vary, the human IgG heavy-chain Fcregion is usually defined to stretch from an amino acid residue atposition Cys226, or from Pro230, to the carboxyl-terminus thereof. TheC-terminal lysine (residue 447 according to the EU numbering system) ofthe Fc region may be removed, for example, during production orpurification of the antibody, or by recombinantly engineering thenucleic acid encoding a heavy chain of the antibody. Accordingly, acomposition of intact antibodies may comprise antibody populations withall K447 residues removed, antibody populations with no K447 residuesremoved, and antibody populations having a mixture of antibodies withand without the K447 residue. Suitable native-sequence Fc regions foruse in the antibodies of the invention include human IgG1, IgG2, IgG3and IgG4.

A “native sequence Fc region” comprises an amino acid sequence identicalto the amino acid sequence of an Fc region found in nature. Nativesequence human Fc regions include a native sequence human IgG1 Fc region(non-A and A allotypes); native sequence human IgG2 Fc region; nativesequence human IgG3 Fc region; and native sequence human IgG4 Fc regionas well as naturally occurring variants thereof.

A “variant Fc region” comprises an amino acid sequence which differsfrom that of a native sequence Fc region by virtue of at least one aminoacid modification, preferably one or more amino acid substitution(s).Preferably, the variant Fc region has at least one amino acidsubstitution compared to a native sequence Fc region or to the Fc regionof a parent polypeptide, e.g. from about one to about ten amino acidsubstitutions, and preferably from about one to about five amino acidsubstitutions in a native sequence Fc region or in the Fc region of theparent polypeptide. The variant Fc region herein will preferably possessat least about 80% homology with a native sequence Fc region and/or withan Fc region of a parent polypeptide, and most preferably at least about90% homology therewith, more preferably at least about 95% homologytherewith.

“Fc receptor” or “FcR” describes a receptor that binds to the Fc regionof an antibody. The preferred FcR is a native sequence human FcR.Moreover, a preferred FcR is one which binds an IgG antibody (a gammareceptor) and includes receptors of the FcγRI, FcγRII, and FcγRIIIsubclasses, including allelic variants and alternatively spliced formsof these receptors, FcγRII receptors include FcγRIIA (an “activatingreceptor”) and FcγRIIB (an “inhibiting receptor”), which have similaramino acid sequences that differ primarily in the cytoplasmic domainsthereof. Activating receptor FcγRIIA contains an immunoreceptortyrosine-based activation motif (“ITAM”) in its cytoplasmic domain.Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-basedinhibition motif (“ITIM”) in its cytoplasmic domain. (see, e.g., M.Daëron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed inRavetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991); Capel et al.,Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med.126: 330-41 (1995). Other FcRs, including those to be identified in thefuture, are encompassed by the term “FcR” herein. FcRs can also increasethe serum half-life of antibodies.

Binding to FcRn in vivo and serum half-life of human FcRn high-affinitybinding polypeptides can be assayed, e.g., in transgenic mice ortransfected human cell lines expressing human FcRn, or in primates towhich the polypeptides having a variant Fc region are administered. WO2004/42072 (Presta) describes antibody variants with improved ordiminished binding to FcRs. See also, e.g., Shields et al., J. Biol.Chem. 9(2):6591-6604 (2001).

As used herein, “percent (%) amino acid sequence identity” and“homology” with respect to a peptide, polypeptide or antibody sequencerefers to the percentage of amino acid residues in a candidate sequencethat are identical with the amino acid residues in the specific peptideor polypeptide sequence, after aligning the sequences and introducinggaps, if necessary, to achieve the maximum percent sequence identity,and not considering any conservative substitutions as part of thesequence identity. Alignment for purposes of determining percent aminoacid sequence identity can be achieved in various ways that are withinthe skill in the art, for instance, using publicly available computersoftware such as BLAST, BLAST-2, ALIGN or MEGALIGN™ (DNASTAR) software.Those skilled in the art can determine appropriate parameters formeasuring alignment, including any algorithms known in the art needed toachieve maximal alignment over the full length of the sequences beingcompared.

An “isolated” nucleic acid molecule encoding an antibody, such as ananti-TREM2 and/or anti-DAP12 antibody of the present disclosure, is anucleic acid molecule that is identified and separated from at least onecontaminant nucleic acid molecule with which it is ordinarily associatedin the environment in which it was produced. Preferably, the isolatednucleic acid is free of association with all components associated withthe production environment. The isolated nucleic acid molecules encodingthe polypeptides and antibodies herein is in a form other than in theform or setting in which it is found in nature. Isolated nucleic acidmolecules therefore are distinguished from nucleic acid encoding thepolypeptides and antibodies herein existing naturally in cells.

The term “vector,” as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid,” which refers to acircular double stranded DNA into which additional DNA segments may beligated. Another type of vector is a phage vector. Another type ofvector is a viral vector, wherein additional DNA segments may be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)can be integrated into the genome of a host cell upon introduction intothe host cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “recombinant expression vectors,” or simply, “expressionvectors.” In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” may be used interchangeably as theplasmid is the most commonly used form of vector.

“Polynucleotide,” or “nucleic acid,” as used interchangeably herein,refer to polymers of nucleotides of any length, and include DNA and RNA.The nucleotides can be deoxyribonucleotides, ribonucleotides, modifiednucleotides or bases, and/or their analogs, or any substrate that can beincorporated into a polymer by DNA or RNA polymerase or by a syntheticreaction. A polynucleotide may comprise modified nucleotides, such asmethylated nucleotides and their analogs. If present, modification tothe nucleotide structure may be imparted before or after assembly of thepolymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may comprise modification(s)made after synthesis, such as conjugation to a label. Other types ofmodifications include, for example, “caps,” substitution of one or moreof the naturally occurring nucleotides with an analog, internucleotidemodifications such as, for example, those with uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoamidates, carbamates,etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example, proteins (e.g., nucleases, toxins, antibodies, signalpeptides, ply-L-lysine, etc.), those with intercalators (e.g., acridine,psoralen, etc.), those containing chelators (e.g., metals, radioactivemetals, boron, oxidative metals, etc.), those containing alkylators,those with modified linkages (e.g., alpha anomeric nucleic acids, etc.),as well as unmodified forms of the polynucleotides(s). Further, any ofthe hydroxyl groups ordinarily present in the sugars may be replaced,for example, by phosphonate groups, phosphate groups, protected bystandard protecting groups, or activated to prepare additional linkagesto additional nucleotides, or may be conjugated to solid or semi-solidsupports. The 5′ and 3′ terminal OH can be phosphorylated or substitutedwith amines or organic capping group moieties of from 1 to 20 carbonatoms. Other hydroxyls may also be derivatized to standard protectinggroups. Polynucleotides can also contain analogous forms of ribose ordeoxyribose sugars that are generally known in the art, including, forexample, 2′-O-methyl-, 2′-O-allyl-, 2′-fluoro- or 2′-azido-ribose,carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such asarabinose, xyloses or lyxoses, pyranose sugars, furanose sugars,sedoheptuloses, acyclic analogs, and basic nucleoside analogs such asmethyl riboside. One or more phosphodiester linkages may be replaced byalternative linking groups. These alternative linking groups include,but are not limited to, embodiments wherein phosphate is replaced byP(O)S (“thioate”), P(S)S (“dithioate”), (O)NR2 (“amidate”), P(O)R,P(O)OR′, CO, or CH2 (“formacetal”), in which each R or R′ isindependently H or substituted or unsubstituted alkyl (1-20 C)optionally containing an ether (—O—) linkage, aryl, alkenyl, cycloalkyl,cycloalkenyl or araldyl. Not all linkages in a polynucleotide need beidentical. The preceding description applies to all polynucleotidesreferred to herein, including RNA and DNA.

A “host cell” includes an individual cell or cell culture that can be orhas been a recipient for vector(s) for incorporation of polynucleotideinserts. Host cells include progeny of a single host cell, and theprogeny may not necessarily be completely identical (in morphology or ingenomic DNA complement) to the original parent cell due to natural,accidental, or deliberate mutation. A host cell includes cellstransfected in vivo with a polynucleotide(s) of this invention.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers that are nontoxic to the cell or mammal beingexposed thereto at the dosages and concentrations employed. Often thephysiologically acceptable carrier is an aqueous pH buffered solution.Examples of physiologically acceptable carriers include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid; low molecular weight (less than about 10 residues)polypeptide; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, arginine or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; salt-forming counterions such assodium; and/or nonionic surfactants such as TWEEN™, polyethylene glycol(PEG), and PLURONICS™.

The term “about” as used herein refers to the usual error range for therespective value readily known to the skilled person in this technicalfield. Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference unless the context clearly indicatesotherwise. For example, reference to an “antibody” is a reference tofrom one to many antibodies, such as molar amounts, and includesequivalents thereof known to those skilled in the art, and so forth.

It is understood that aspect and embodiments of the invention describedherein include “comprising,” “consisting,” and “consisting essentiallyof” aspects and embodiments.

Overview

The present disclosure relates to anti-TREM2 antibodies and/oranti-DAP12 antibodies with one or more agonist or antagonist activities;methods of making and using such antibodies; pharmaceutical compositionscontaining such antibodies; nucleic acids encoding such antibodies; andhost cells containing nucleic acids encoding such antibodies.

In some embodiments, and without wishing to be bound by theory, it isbelieved that the agonistic activities of the anti-TREM2 and/oranti-DAP12 antibodies of the present disclosure are due, at least inpart, to the ability of the antibodies to induce or retain TREM2receptor clustering on the surface of cells. In some embodiments, it isbelieved that anti-TREM2 and/or anti-DAP12 antibodies can induce orretain TREM2 receptor clustering in vivo by not only bindingspecifically to TREM2, but by also binding to Fc receptors on adjacentcells, which leads to antibody aggregation that in turn aggregatesTREM2. Advantageously, certain immunoglobulin isotypes, includingwithout limitation, IgG2 and IgM, have an intrinsic ability to induce orretain clustering of target antigens (e.g., TREM2) without binding Fcreceptors on adjacent cells. In some embodiments, agonistic TREM2activities can be determined or tested in vitro by any of severaltechniques disclosed herein (see, e.g., Examples 23-26, 34-37, 41-44,52-55, and 67-68), including, without limitation, plate-bindingfull-length anti-TREM2 antibodies to increase the density of antibodiesexposed to TREM2 and cross-linking anti-TREM2 antibodies.

Accordingly, certain aspects of the present disclosure are based, atleast in part, on the identification of anti-TREM2 and/or anti-DAP12antibodies that are capable of binding to both human and mouse TREM2with high affinity (see, e.g., Examples land 40); that compete withTREM2-ligand for binding to the ligand-binding site on human and mouseTREM2 (see, e.g., Examples 26 and 43); and that exhibit one or moreagonistic TREM2 activities, including, without limitation, induction ofCD83⁺CD86⁺ dendritic cells (see, e.g., Examples 23 and 41), induction ofthe TREM2 downstream signaling molecule Syk in macrophages and dendriticcells (see, e.g., Examples 24 and 42), induction of the TREM2 signalingadaptor molecule DAP12 in macrophages (see, e.g., Examples 25 and 44),induction of cell survival of innate immune cells, such as macrophages(see, e.g., Examples 34 and 52), and activation of TREM2-dependent geneexpression (see, e.g., Examples 36, 38, 54, 56, and 68).

Further aspects of the present disclosure are based, at least in part,on the surprising discovery that the anti-TREM2 and/or anti-DAP12antibodies of the present disclosure can also induce antagonisticactivities when the antibody is produced or otherwise formatted suchthat it is incapable of inducing or retaining TREM2 receptor clustering.In some embodiments, anti-TREM2 and/or anti-DAP12 antibodies of thepresent disclosure exhibit one or more antagonistic TREM2 activities,including, without limitation, inhibition of cell survival of innateimmune cells ((see, e.g., Examples 35 and 53), and inhibition ofTREM2-dependent gene expression (see, e.g., Examples 37, 55, and 67).

TREM2 Proteins

In one aspect, the invention provides antibodies that bind to a TREM2protein of the present disclosure and modulate one or more TREM2activities after binding to a TREM2 protein expressed in a cell.

TREM2 proteins of the present disclosure include, without limitation, amammalian TREM2 protein, human TREM2 protein (Uniprot Accession No.Q9NZC2), mouse TREM2 protein (Uniprot Accession No. Q99NH8), rat TREM2protein (Uniprot Accession No. D3ZZ89), Rhesus monkey TREM2 protein(Uniprot Accession No. F6QVF2), bovine TREM2 protein (Uniprot AccessionNo. Q05B59), equine TREM2 protein (Uniprot Accession No. F7D6L0), pigTREM2 protein (Uniprot Accession No. H2EZZ3), and dog TREM2 protein(Uniprot Accession No. E2RP46). As used herein “TREM2 protein” refers toboth wild-type sequences and naturally occurring variant sequences.

Triggering receptor expressed on myeloid cells-2 (TREM2) is variouslyreferred to as TREM-2, Trem2a, Trem2b, Trem2c, triggering receptorexpressed on myeloid cells-2a, and triggering receptor expressed onmonocytes-2. TREM2 is a 230 amino acid membrane protein. TREM2 is animmunoglobulin-like receptor primarily expressed on myeloid lineagecells, including without limitation, macrophages, dendritic cells,osteoclasts, microglia, monocytes, Langerhans cells of skin, and Kupffercells. In some embodiments, TREM2 forms a receptor-signaling complexwith DAP12. In some embodiments, TREM2 phosphorylates and signalsthrough DAP12 (an ITAM domain adaptor protein). In some embodimentsTREM2 signaling results in the downstream activation of PI3K or otherintracellular signals. On Myeloid cells, Toll-like receptor (TLR)signals are important for the activation of TREM2 activities, e.g., inthe context of an infection response. TLRs also play a key role in thepathological inflammatory response, e.g., TLRs expressed in macrophagesand dendritic cells.

In some embodiments, an example of a human TREM2 amino acid sequence isset forth below as SEQ ID NO: 1:

        10         20         30         40 MEPLRLLILL FVTELSGAHNTTVFQGVAGQ SLQVSCPYDS         50         60         70         80MKHWGRRKAW CRQLGEKGPC QRVVSTHNLW LLSFLRRWNG        90        100        110        120 STAITDDTLG GTLTITLRNLQPHDAGLYQC QSLHGSEADT        130        140        150        160LRKVLVEVLA DPLDHRDAGD LWFPGESESF EDAHVEHSIS       170        180        190        200 RSLLEGEIPF PPTSILLLLACIFLIKILAA SALWAAAWHG        210        220        230 QKPGTHPPSELDCGHDPGYQ LQTLPGLRDT

In some embodiments, the human TREM2 is a preprotein that includes asignal peptide. In some embodiments, the human TREM2 is a matureprotein. In some embodiments, the mature TREM2 protein does not includea signal peptide. In some embodiments, the mature TREM2 protein isexpressed on a cell. In some embodiments, TREM2 contains a signalpeptide located at amino acid residues 1-18 of human TREM2 (SEQ ID NO:1); an extracellular immunoglobulin-like variable-type (IgV) domainlocated at amino acid residues 29-112 of human TREM2 (SEQ ID NO: 1);additional extracellular sequences located at amino acid residues113-174 of human TREM2 (SEQ ID NO: 1); a transmembrane domain located atamino acid residues 175-195 of human TREM2 (SEQ ID NO: 1); and anintracellular domain located at amino acid residues 196-230 of humanTREM2 (SEQ ID NO: 1).

The transmembrane domain of human TREM2 contains a lysine at amino acidresidue 186 that can interact with an aspartic acid in DAP12, which is akey adaptor protein that transduces signaling from TREM2, TREM1, andother related IgV family members.

Homologues of human TREM2 include, without limitation, the naturalkiller (NK) cell receptor NK-p44 (NCTR2), the polymeric immunoglobulinreceptor (pIgR), CD300E, CD300A, CD300C, and TREML1/TLT1. In someembodiments, NCTR2 has similarity with TREM2 within the IgV domain.

DAP12 Proteins

In one aspect, the invention provides antibodies that bind to a DAP12protein of the present disclosure and modulate one or more DAP12activities after binding to a DAP12 protein expressed in a cell.

DAP12 proteins of the present disclosure include, without limitation, amammalian DAP12 protein, human DAP12 protein (Uniprot Accession No.043914), mouse DAP12 protein (Uniprot Accession No. 054885), rat DAP12protein (Uniprot Accession No. Q6X9T7), Rhesus monkey DAP12 protein(Uniprot Accession No. Q8WNQ8), bovine DAP12 protein (Uniprot AccessionNo. Q95J80), and pig DAP12 protein (Uniprot Accession No. Q9TU45). Asused herein “DAP12 protein” refers to both wild-type sequences andnaturally occurring variant sequences.

DNAX-activation protein 12 (DAP12) is variously referred to asKiller-activating receptor-associated protein, KAR-associated protein(KARAP), PLOSL, PLO-SL, TYRO protein, and tyrosine kinase-bindingprotein. DAP12 is a 113 amino acid membrane protein. In someembodiments, DAP12 functions as a transmembrane signaling polypeptide,which contains an immunoreceptor tyrosine-based activation motif (ITAM)in its cytoplasmic domain. It may associate with the killer-cellinhibitory receptor (KIR) family of membrane glycoproteins and may actas an activating signal transduction element. In other embodiments, theDAP12 protein may bind zeta-chain (TCR) associated protein kinase 70 kDa(ZAP-70) and spleen tyrosine kinase (SYK), and play a role in signaltransduction, bone modeling, brain myelination, and inflammation.

Mutations within the DAP12-encoding gene have been associated withpolycystic lipomembranous osteodysplasia with sclerosingleukoencephalopathy (PLOSL), also known as Nasu-Hakola disease. Withoutwishing to be bound by theory, it is believed that the DAP12 receptor isTREM2, which also causes PLOSL. Multiple alternative transcript variantsencoding distinct isoforms of DAP12 have been identified. DAP12non-covalently associates with activating receptors of the CD300 family.Cross-linking of CD300-TYROBP/DAP12 complexes results in cellularactivation, such as neutrophil activation mediated by integrin. DAP12 isa homodimer; disulfide-linked protein. In some embodiments, DAP12interacts with SIRPB1, TREM1, CLECSF5, SIGLEC14, CD300LB, CD300E, andCD300D by similarity and via ITAM domain, as well as with SYK via SH2domain. In other embodiments, DAP12 activates SYK, which mediatesneutrophils and macrophages integrin-mediated activation. In otherembodiments, DAP12 interacts with KLRC2 and KIR2DS3.

In some embodiments, an example of a human DAP12 amino acid sequence isset forth below as SEQ ID NO: 2:

        10         20         30         40 MGGLEPCSRL LLLPLLLAVSGLRPVQAQAQ SDCSCSTVSP         50         60         70         80GVLAGIVMGD LVLTVLIALA VYFLGRLVPR GRGAAEAATR        90        100        110 KQRITETESP YQELQGQRSD VYSDLNTQRP YYK

In some embodiments, the human DAP12 is a preprotein that includes asignal peptide. In some embodiments, the human DAP12 is a matureprotein. In some embodiments, the mature DAP12 protein does not includea signal peptide. In some embodiments, the mature DAP12 protein isexpressed on a cell. DAP12 is a single-pass type I membrane protein. Itcontains an extracellular domain located at amino acid residues 22-40 ofhuman DAP12 (SEQ ID NO: 2); a transmembrane domain located at amino acidresidues 41-61 of human DAP12 (SEQ ID NO: 2); and an intracellulardomain located at amino acid residues 62-113 of human DAP12 (SEQ ID NO:2). The immunoreceptor tyrosine-based activation motif (ITAM) domain islocated at amino acid residues 80-118 of human DAP12 (SEQ ID NO: 2).

In some embodiments, an aspartic acid residue in DAP12 interacts withthe transmembrane domain of human TREM2 containing a lysine at aminoacid residue 186, and transduces signaling from TREM2, TREM1, and otherrelated IgV family member proteins.

Anti-TREM2 and Anti-DAP12 Antibodies

Certain aspects of the present disclosure relate to antibodies thatspecifically bind to TREM2 and/or DAP12. In some embodiments, antibodiesof the present disclosure bind a mature TREM2 protein and/or DAP12protein. In some embodiments, antibodies of the present disclosure binda mature TREM2 protein and/or DAP12 protein, wherein the mature TREM2protein and/or DAP12 protein is expressed on a cell. In someembodiments, antibodies of the present disclosure bind a TREM2 proteinand/or DAP12 protein expressed on one or more human cells selected fromhuman dendritic cells, human macrophages, human monocytes, humanosteoclasts, human Langerhans cells of skin, human Kupffer cells, humanmicroglia, and any combinations thereof. In some embodiments, antibodiesof the present disclosure are agonist antibodies. In some embodiments,antibodies of the present disclosure are inert antibodies. In someembodiments, antibodies of the present disclosure are antagonistantibodies.

Agonist Antibodies

Anti-TREM2 and/or anti-DAP12 antibodies of the present disclosuregenerally bind to one or more TREM2 proteins and/or DAP12 proteinsexpressed in a cell. One class of antibodies is agonist antibodies. Forexample, the TREM2 receptor is thought to require clustering on the cellsurface in order to transduce a signal. Thus agonist antibodies may haveunique features to stimulate, for example, the TREM2 receptor. Forexample, they may have the correct epitope specificity that iscompatible with receptor activation, as well as the ability to induce orretain receptor clustering on the cell surface.

In vivo, antibodies may cluster receptors by multiple potentialmechanisms. Some isotypes of human antibodies such as IgG2 have, due totheir unique structure, an intrinsic ability to cluster receptors, orretain receptors in a clustered configuration, thereby activatingreceptors such as TREM2 without binding to an Fc receptor (e.g., Whiteet al., (2015) Cancer Cell 27, 138-148).

Other antibodies cluster receptors (e.g., TREM2) by binding to Fcgreceptors on adjacent cells. Binding of the constant IgG Fc part of theantibody to Fcg receptors leads to aggregation of the antibodies, andthe antibodies in turn aggregate the receptors to which they bindthrough their variable region (Chu et al (2008) Mol Immunol,45:3926-3933; and Wilson et al., (2011) Cancer Cell 19, 101-113).Binding to the inhibitory Fcg receptor FcgR (FcgRIIB) that does notelicit cytokine secretion, oxidative burst, increased phagocytosis, andenhanced antibody-dependent, cell-mediated cytotoxicity (ADCC) is oftena preferred way to cluster antibodies in vivo, since binding to FcgRIIBis not associated with immune adverse effects.

Other mechanisms may also be used to cluster receptors (e.g., TREM2).For example, antibody fragments (e.g., Fab fragments) that arecross-linked together may be used to cluster receptors (e.g., TREM2) ina manner similar to antibodies with Fc regions that bind Fcg receptors,as described above. Without wishing to be bound to theory, it is thoughtthat cross-linked antibody fragments (e.g., Fab fragments) may functionas agonist antibodies if they induce receptor clustering on the cellsurface and bind an appropriate epitope on the target (e.g., TREM2).

Therefore, in some embodiments, antibodies that bind a TREM2 proteinand/or a DAP12 protein may include agonist antibodies that due to theirepitope specificity bind TREM2 and/or DAP12 and activate one or moreTREM2 and/or DAP12 activities. Without wishing to be bound to theory,such antibodies may bind to the ligand-binding site on the targetantigen (e.g., TREM2 and/or DAP12) and mimic the action of a naturalligand, or stimulate the target antigen to transduce signal by bindingto one or more domains that are not the ligand-binding sites. Suchantibodies would not interfere with ligand binding and may actadditively or synergistically with the natural ligands.

In some embodiments, an antibody of the present disclosure is an agonistantibody that induces one or more TREM2 activities, one or more DAP12activities, or one or more TREM2 activities and one or more DAP12activities. In some embodiments the antibody induces one or moreactivities of TREM2 and/or DAP12 after binding to a TREM2 and/or DAP12protein that is expressed in a cell. In certain embodiments, the one ormore TREM2 activities, the one or more DAP12 activities, or both areselected from TREM2 binding to DAP12; DAP12 binding to TREM2; DAP12phosphorylation; TREM2 phosphorylation; PI3K activation; increasedexpression of one or more anti-inflammatory cytokines, increasedexpression of one or more anti-inflammatory mediators (e.g., cytokines)selected from IL-12p70, IL-6, and IL-10; reduced expression of one ormore pro-inflammatory cytokines; reduced expression of one or morepro-inflammatory mediators selected from the group consisting of IFN-a4,IFN-b, IL-6, IL-12 p70, IL-1β, TNG, TNF-α, IL-10, IL-8, CRP, TGF-betamembers of the chemokine protein families, IL-20 family members, IL-33,LIF, IFN-gamma, OSM, CNTF, TGF-beta, GM-CSF, IL-11, IL-12, IL-17, IL-18,mCP-1, and CRP; reduced expression of TNF-α; reduced expression of IL-6;extracellular signal-regulated kinase (ERK) phosphorylation; increasedexpression of C-C chemokine receptor 7 (CCR7); induction of microglialcell chemotaxis toward CCL19 and CCL21 expressing cells; an enhancement,normalization, or both of the ability of bone marrow-derived dendriticcells to induce antigen-specific T-cell proliferation; induction ofosteoclast production, increased rate of osteoclastogenesis, or both;increasing the survival and/or function of one or more of macrophages,microglial cells, M1 macrophages and/or microglial cells, activated M1macrophages and/or microglial cells, M2 macrophages and/or microglialcells, monocytes, osteoclasts, Langerhans cells of skin, and Kupffercells; induction of one or more types of clearance selected fromapoptotic neuron clearance, nerve tissue debris clearance, non-nervetissue debris clearance, bacteria or other foreign body clearance,disease-causing protein clearance, disease-causing peptide clearance,and disease-causing nucleic acid clearance; induction of phagocytosis ofone or more of apoptotic neurons, nerve tissue debris, non-nerve tissuedebris, bacteria, other foreign bodies, disease-causing proteins,disease-causing proteins, disease-causing peptides, or disease-causingnucleic acids (e.g., antisense GGCCCC (G2C4) repeat-expansion RNA);normalization of disrupted TREM2/DAP12-dependent gene expression;recruitment of Syk, ZAP70, or both to a DAP12/TREM2 complex; Sykphosphorylation; increased expression of CD83 and/or CD86 on dendriticcells, macrophages, monocytes, and/or microglia; reduced secretion ofone or more inflammatory cytokines; reduced secretion of one or moreinflammatory cytokines selected from TNF-α, IL-10, IL-6, MCP-1, FN-a4,IFN-b, IL-1β. IL-8, CRP, TGF-beta members of the chemokine proteinfamilies, IL-20 family members, IL-33, LIF, IFN-gamma, OSM, CNTF,TGF-beta, GM-CSF, IL-11, IL-12, IL-17, and IL-18; reduced expression ofone or more inflammatory receptors; increasing phagocytosis bymacrophages, dendritic cells, monocytes, and/or microglia underconditions of reduced levels of MCSF; decreasing phagocytosis bymacrophages, dendritic cells, monocytes, and/or microglia in thepresence of normal levels of MCSF; increasing activity of one or moreTREM2-dependent genes (e.g., transcription factors of the nuclear factorof activated T-cells (NFAT) family of transcription factors).

An antibody dependent on binding to FcgR receptor to activate targetedreceptors may lose its agonist activity if engineered to eliminate FcgRbinding (see, e.g., Wilson et al., (2011) Cancer Cell 19, 101-113;Armour at al., (2003) Immunology 40 (2003) 585-593); and White et al.,(2015) Cancer Cell 27, 138-148). As such, it is thought that an antibodyof the present disclosure with the correct epitope specificity can be anagonist antibody and activate the target antigen, with minimal adverseeffects, when the antibody has an Fc domain from a human IgG2 isotype(CH1 and hinge region) or another type of Fc domain that is capable ofpreferentially binding the inhibitory FcgRIIB r receptors, or avariation thereof.

Exemplary agonist antibody Fc isotypes and modifications are provided inTable 2 below. In some embodiments, the agonist antibody has an Fcisotype listed in Table 2 below.

TABLE 2 Exemplary anti-TREM2 agonist antibody Fc isotypes Fc IsotypeMutation (EU or Kabat numbering scheme) IgG1 N297A IgG1 D265A and N297AIgG1 L234A and L235A L234A and G237A L234A and L235A and G237A IgG2V234A and G237A IgG4 L235A and G237A and E318A IgG4 S228P and L236EIgG2/4 hybrid IgG2 aa 118-260 and IgG4 aa 261 to 447 IgG2 H268Q andV309L; and A330S and P331S IgG1 C226S and C229S and E233P and L234V andL235A IgG1 L234F and L235E and P331S IgG2 C232S or C233S IgG2 A330S andP331S IgG1 S267E, and L328F S267E alone IgG2 S267E and L328F IgG4 S267Eand L328F IgG2 WT HC with Kappa (light chain) LC HC C127S with Kappa LCKappa LC C214S Kappa LC C214S and HC C233S Kappa LC C214S and HC C232SAny of the above listed mutations together with P330S and P331Smutations F(ab′)2 fragment of WT IgG1 and any of the above listedmutations IgG1 Substitute the Constant Heavy 1 (CH1) and hinge region ofIgG1 With CH1 and hinge region of IGg2 ASTKGPSVFP LAPCSRSTSE STAALGCLVKDYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSNFGTQT YTCNVDHKPSNTKVDKTVER KCCVECPPCP (SEQ ID NO: 397) With a Kappa LC IgG1 Any of theabove listed mutations together with A330L and/or L234F and/or L235Eand/or P331S IgG1, IgG2, or IgG4 Any of the above listed mutationstogether with M252Y and/or S254T and/or T256E Mouse IgG1 For mousedisease models IgG4 WT

In addition to the isotypes described in Table 2, and without wishing tobe bound to theory, it is thought that antibodies with human IgG1 orIgG3 isotypes and mutants thereof (e.g. Strohl (2009) Current Opinion inBiotechnology 2009, 20:685-691) that bind the activating Fcg ReceptorsI, IIA, IIC, IIIA, IIIB in human and/or Fcg Receptors I, III and IV inmouse, may also act as agonist antibodies in vivo but may be associatedwith adverse effects related to ADCC. However, such Fcg receptors appearto be less available for antibody binding in vivo, as compared to theInhibitory Fcg receptor FcgRIIB (see, e.g., White, et al., (2013) CancerImmunol. Immunother. 62, 941-948; and Li et al., (2011) IScience333(6045):1030-1034.).

In some embodiments, the agonist antibody is of the IgG class, the IgMclass, or the IgA class. In some embodiments, the agonist antibody hasan IgG1, IgG2, IgG3, or IgG4 isotype.

In certain embodiments, the agonist antibody has an IgG2 isotype. Insome embodiments, the agonist antibody contains a human IgG2 constantregion. In some embodiments, the human IgG2 constant region includes anFc region. In some embodiments, the agonist antibody induces the one ormore TREM2 activities, the DAP12 activities, or both independently ofbinding to an Fc receptor. In some embodiments, the agonist antibodybinds an inhibitory Fc receptor. In certain embodiments, the inhibitoryFc receptor is inhibitory Fc-gamma receptor IIB (FcγIIB). In someembodiments, the Fc region contains one or more modifications. Forexample, in some embodiments, the Fc region contains one or more aminoacid substitutions (e.g., relative to a wild-type Fc region of the sameisotype). In some embodiments, the one or more amino acid substitutionsare selected from V234A (Alegre et al., (1994) Transplantation57:1537-1543. 31; Xu et al., (2000) Cell Immunol, 200:16-26), G237A(Cole et al. (1999) Transplantation, 68:563-571), H268Q, V309L, A330S,P331S (US 2007/0148167; Armour et al. (1999) Eur J Immunol 29:2613-2624; Armour et al. (2000) The Haematology Journal 1(Suppl.1):27;Armour et al. (2000) The Haematology Journal 1(Suppl.1):27), C232S,and/or C233S (White et al. (2015) Cancer Cell 27, 138-148), S267E, L328F(Chu et al., (2008) Mol Immunol, 45:3926-3933), M252Y, S254T, and/orT256E, where the amino acid position is according to the EU or Kabatnumbering convention.

In some embodiments, the agonist antibody has an IgG2 isotype with aheavy chain constant domain that contains a C127S amino acidsubstitution, where the amino acid position is according to the EU orKabat numbering convention (White et al., (2015) Cancer Cell 27,138-148; Lightle et al., (2010) PROTEIN SCIENCE 19:753-762; andWO2008079246).

In some embodiments, the agonist antibody has an IgG2 isotype with aKappa light chain constant domain that contains a C214S amino acidsubstitution, where the amino acid position is according to the EU orKabat numbering convention (White et al., (2015) Cancer Cell 27,138-148; Lightle et al., (2010) PROTEIN SCIENCE 19:753-762; andWO2008079246).

In certain embodiments, the agonist antibody has an IgG1 isotype. Insome embodiments, the agonist antibody contains a mouse IgG1 constantregion. In some embodiments, the agonist antibody contains a human IgG1constant region. In some embodiments, the human IgG1 constant regionincludes an Fc region. In some embodiments, the agonist antibody bindsan inhibitory Fc receptor. In certain embodiments, the inhibitory Fcreceptor is inhibitory Fc-gamma receptor IIB (FcγIIB). In someembodiments, the Fc region contains one or more modifications. Forexample, in some embodiments, the Fc region contains one or more aminoacid substitutions (e.g., relative to a wild-type Fc region of the sameisotype). In some embodiments, the one or more amino acid substitutionsare selected from N297A (Bolt S et al. (1993) Eur J Immunol 23:403-411),D265A (Shields et al. (2001) R. J. Biol. Chem. 276, 6591-6604), L234A,L235A (Hutchins et al. (1995) Proc Natl Acad Sci USA, 92:11980-11984;Alegre et al., (1994) Transplantation 57:1537-1543. 31; Xu et al.,(2000) Cell Immunol, 200:16-26), G237A (Alegre et al. (1994)Transplantation 57:1537-1543. 31; Xu et al. (2000) Cell Immunol,200:16-26), C226S, C229S, E233P, L234V, L234F, L235E (McEarchern et al.,(2007) Blood, 109:1185-1192), P331S (Sazinsky et al., (2008) Proc NatlAcad Sci USA 2008, 105:20167-20172), S267E, L328F, A330L, M252Y, S254T,and/or T256E, where the amino acid position is according to the EU orKabat numbering convention.

In some embodiments, the antibody includes an IgG2 isotype heavy chainconstant domain 1(CH1) and hinge region (White et al., (2015) CancerCell 27, 138-148). In certain embodiments, the IgG2 isotype CH1 andhinge region contain the amino acid sequence ofASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSNFGTQT YTCNVDHKPS NTKVDKTVERKCCVECPPCP (SEQ ID NO: 397). In someembodiments, the antibody Fc region contains a S267E amino acidsubstitution, a L328F amino acid substitution, or both, and/or a N297Aor N297Q amino acid substitution, where the amino acid position isaccording to the EU or Kabat numbering convention.

In certain embodiments, the agonist antibody has an IgG4 isotype. Insome embodiments, the agonist antibody contains a human IgG4 constantregion. In some embodiments, the human IgG4 constant region includes anFc region. In some embodiments, the agonist antibody binds an inhibitoryFc receptor. In certain embodiments, the inhibitory Fc receptor isinhibitory Fc-gamma receptor IIB (FcγIIB). In some embodiments, the Fcregion contains one or more modifications. For example, in someembodiments, the Fc region contains one or more amino acid substitutions(e.g., relative to a wild-type Fc region of the same isotype). In someembodiments, the one or more amino acid substitutions are selected fromL235A, G237A, S228P, L236E (Reddy et al., (2000) J Immunol,164:1925-1933), S267E, E318A, L328F, M252Y, S254T, and/or T256E, wherethe amino acid position is according to the EU or Kabat numberingconvention.

In certain embodiments, the agonist antibody has a hybrid IgG2/4isotype. In some embodiments, the agonist antibody includes an aminoacid sequence containing amino acids 118 to 260 according to Kabatnumbering convention of human IgG2 and amino acids 261 to 447 accordingto EU or Kabat numbering convention of human IgG4 (WO 1997/11971; WO2007/106585).

In certain embodiments, the antibody contains a mouse IgG4 constantregion (Bartholomaeus, et al. (2014). J. Immunol. 192, 2091-2098).

In some embodiments, the Fc region further contains one or moreadditional amino acid substitutions selected from the group consistingof A330L, L234F, L235E, and/or P331 S, where the amino acid position isaccording to the EU or Kabat numbering convention.

Inert Antibodies

Another class of antibodies of the present disclosure includes inertantibodies. As used herein, “inert” antibodies refer to antibodies thatspecifically bind their target antigen but do not modulate (e.g.,decrease/inhibit or activate/induce) antigen function. For example, inthe case of TREM2, inert antibodies do not modulate ligand bindingand/or TREM2 activities. Without wishing to be bound to theory, it isthought that antibodies that do not have the ability to cluster TREM2 onthe cell surface may be inert antibodies even if they have an epitopespecificity that is compatible with receptor activation.

In some embodiments, antibodies that bind a TREM2 protein and/or a DAP12protein may include antibodies that bind TREM2 and/or DAP2 but, due totheir epitope specificity, do not modulate protein function. Suchfunctionally inert antibodies can be used as cargo to transport toxinsas described for the CD33 antibody Gemtuzumab zogamicin, (marketed asMylotarg) which is conjugated to the cytotoxic agent from the class ofcalichcamicins and is used to target and kill acute myelogenous leukemiatumors (Naito et al., (2000), Leukemia, 14, 1436-1443; Ricart (2011)Clin Cancer Res 17; 6417-6436; Hamann et al., (2002) Journal:Bioconjugate Chemistry, 13, 47-58; and Beitz et al., (2001) Clin CancerRes 7; 1490-6.). Therefore, in some embodiments, antibodies of thepresent disclosure are inert antibodies that bind TREM2 and/or DAP12 butare incapable of inducing one or more TREM2 activities (e.g., a TREM2activity described herein) and/or DAP12 activities (e.g., a DAP12activity described herein).

Exemplary inert antibody Fc isotypes and modifications are provided inTable 3 below. In some embodiments, the inert antibody has an Fc isotypelisted in Table 3 below.

Antagonist Antibodies

A third class of antibodies of the present disclosure includesantagonist antibodies. In some embodiments, antibodies that bind a TREM2protein and/or a DAP12 protein may include antagonist antibodies thatbind TREM2 and/or DAP12 and inhibit one or more TREM2 activities and/orDAP12 activities, either by preventing interaction between TREM2 and/orDAP12 and its ligand(s), or by preventing the transduction of signalfrom the extracellular domain of TREM2 and/or DAP12 into the cellcytoplasm in the presence of ligand. In some embodiments, antagonistantibodies of the present disclosure may have the epitope specificity ofan agonist antibody of the present disclosure, but have an Fe domainthat is not capable of binding Fcg receptors and thus is unable to, forexample, cluster DAP12 and/or the TREM2 receptor.

In some embodiments, an antibody of the present disclosure is anantagonist antibody. In some embodiments, the antagonist antibodyinhibits one or more TREM2 and/or DAP12 activities. In some embodiments,the antagonist antibody decreases activity of one or moreTREM2-dependent genes. In some embodiments, the one or moreTREM2-dependent genes include, without limitation, one or more nuclearfactor of activated T-cells (NFAT) transcription factors. In someembodiments, the antagonist antibody decreases the survival ofmacrophages, microglial cells, M1 macrophages, M1 microglial cells, M2macrophages, M2 microglial cells, osteoclasts, Langerhans cells of skin,Kupffer cells, and/or dendritic cell. In some embodiments, theantagonist antibody inhibits interaction between TREM2 and/or DAP12 andone or more TREM2 and/or DAP12 ligands. In some embodiments, theantagonist antibody inhibits TREM2 and/or DAP12 signal transduction. Insome embodiments, the antagonist antibody inhibits interaction betweenTREM2 and/or DAP12 and one or more TREM2 and/or DAP12 ligands andinhibits TREM2 and/or DAP12 signal transduction.

In some embodiments, antibody cross-linking is required for agonistantibody function. Antibody cross-linking can occur through binding to asecondary antibody in vitro or through binding to Fc receptors in vivo.For example, antagonistic antibodies can be converted to agonisticantibodies via biotin/streptavidin cross-linking or secondary antibodybinding in vitro (see for example Gravestein et al., (1996) J. Exp. Med.184:675-685; Gravestein et al., (1994) International Immunol.7:551-557). Agonistic antibodies may exert their activity by mimickingthe biological activity of the receptor ligand or by enhancing receptoraggregation, thereby activating receptor signaling. In some embodiments,the absence of antibody cross-linking is required for antagonisticactivity. Antagonistic antibodies may exert their activity by blockingreceptor-ligand interactions.

Exemplary antagonist antibody Fc isotypes and modifications are providedin Table 3 below. In some embodiments, the antagonist antibody has an Fcisotype listed in Table 3 below.

Inert and Antagonist Antibody Fc Isotypes

In some embodiments, inert and/or antagonist anti-TREM antibodies of thepresent disclosure include one or more of the Fc isotypes andmodifications listed in Table 3.

TABLE 3 Exemplary inert and antagonist anti-TREM2 antibody Fc isotypesMutation (EU or Kabat Fc Isotype numbering scheme) IgG1 N297Aor N297QIgG1 D265A and N297A IgG1 L234A and L235A IgG2 V234A and G237A IgG4L235A and G237A and E318A E233P and/or F234V N297Aor N297Q IgG4 S228Pand L236E S241P S241P and L248E IgG2 H268Q and V309L and A330S and P331SIgG1 C220S and C226S and C229S and P238S IgG1 C226S and C229S and E233Pand L234V, and L235A IgG1 E233P and L234V and L235A and G236-deletedP238A D265A N297A A327Q or A327G P329A IgG1 K322A and L234A and L235AIgG1 L234Fand L235E and P331S IgG1 or IgG4 T394D IgG2 C232S or C233SN297Aor N297Q IgG1, IgG2, delta a, b, c, ab, ac, g or IgG4 modificationsIgG1 Any of the above listed mutations together with A330L or L234Fand/or L235E and/or P331S IgG1, IgG2, Any of the above listed mutationsor IgG4 together with M252Y and/or S254T and/or T256E

In certain embodiments, the antibody has an IgG1 isotype. In someembodiments, the antibody contains a mouse IgG1 constant region. In someembodiments, the antibody contains a human IgG1 constant region. In someembodiments, the human IgG1 constant region includes an Fc region. Insome embodiments, the Fc region contains one or more modifications. Forexample, in some embodiments, the Fc region contains one or more aminoacid substitutions (e.g., relative to a wild-type Fc region of the sameisotype). In some embodiments, the one or more amino acid substitutionsare selected from N297A, N297Q (Bolt S et al. (1993) Eur J Immunol23:403-411), D265A, L234A, L235A (McEarchern et al., (2007) Blood,109:1185-1192), C226S, C229S (McEarchern et al., (2007) Blood,109:1185-1192), P238S (Davis et al., (2007) J Rheumatol, 34:2204-2210),E233P, L234V (McEarchern et al., (2007) Blood, 109:1185-1192), P238A,A327Q, A327G, P329A (Shields R L. et al., (2001) J Biol Chem.276(9):6591-604), K322A, L234F, L235E (Hezareh, et al., (2001) J Virol75, 12161-12168; Oganesyan et al., (2008). Acta Crystallographica 64,700-704), P331S (Oganesyan et al., (2008) Acta Crystallographica 64,700-704), T394D (Wilkinson et al. (2013) MAbs 5(3): 406-417), A330L,M252Y, S254T, and/or T256E, where the amino acid position is accordingto the EU or Kabat numbering convention. In certain embodiments, the Fcregion further includes an amino acid deletion at a positioncorresponding to glycine 236 according to the EU or Kabat numberingconvention.

In some embodiments, the antibody has an IgG1 isotype with a heavy chainconstant region that contains a C220S amino acid substitution accordingto the EU or Kabat numbering convention.

In some embodiments, the Fc region further contains one or moreadditional amino acid substitutions selected from A330L, L234F; L235E,and/or P331S according to EU or Kabat numbering convention.

In certain embodiments, the antibody has an IgG2 isotype. In someembodiments, the antibody contains a human IgG2 constant region. In someembodiments, the human IgG2 constant region includes an Fc region. Insome embodiments, the Fc region contains one or more modifications. Forexample, in some embodiments, the Fc region contains one or more aminoacid substitutions (e.g., relative to a wild-type Fc region of the sameisotype). In some embodiments, the one or more amino acid substitutionsare selected from V234A, G237A, H268E, V309L, N297A, N297Q, A330S,P331S, C232S, C233S, M252Y, S254T, and/or T256E, where the amino acidposition is according to the EU or Kabat numbering convention.

In certain embodiments, the antibody has an IgG4 isotype. In someembodiments, the antibody contains a human IgG4 constant region. In someembodiments, the human IgG4 constant region includes an Fc region. Insome embodiments, the Fc region contains one or more modifications. Forexample, in some embodiments, the Fc region contains one or more aminoacid substitutions (e.g., relative to a wild-type Fc region of the sameisotype). In some embodiments, the one or more amino acid substitutionsare selected from E233P, F234V, L235A, G237A, E318A (Hutchins et al.(1995) Proc Natl Acad Sci USA, 92:11980-11984), S228P, L236E, S241P,L248E (Reddy et al., (2000) J Immunol, 164:1925-1933; Angal et al.,(1993) Mol Immunol. 30(1):105-8; U.S. Pat. No. 8,614,299 B2), T394D,M252Y, S254T, T256E, N297A, and/or N297Q, where the amino acid positionis according to the EU or Kabat numbering convention.

In some embodiments, the Fc region further contains one or moreadditional amino acid substitutions selected from a M252Y, S254T, and/orT256E, where the amino acid position is according to the EU or Kabatnumbering convention.

Further IgG Mutations

In some embodiments, one or more of the IgG1 variants described hereinmay be combined with an A330L mutation (Lazar et al., (2006) Proc NatlAcad Sci USA, 103:4005-4010), or one or more of L234F, L235E, and/orP331S mutations (Sazinsky et al., (2008) Proc Natl Acad Sci USA,105:20167-20172), where the amino acid position is according to the EUor Kabat numbering convention, to eliminate complement activation. Insome embodiments, the IgG variants described herein may be combined withone or more mutations to enhance the antibody half-life in human serum(e.g. M252Y, S254T, T256E mutations according to the EU or Kabatnumbering convention) (Dall'Acqua et al., (2006) J Biol Chem,281:23514-23524; and Strohl e al., (2009) Current Opinion inBiotechnology, 20:685-691).

In some embodiments, an IgG4 variant of the present disclosure may becombined with an S228P mutation according to the EU or Kabat numberingconvention (Angal et al., (1993) Mol Immunol, 30:105-108) and/or withone or more mutations described in Peters et al., (2012) J Biol Chem.13; 287(29):24525-33) to enhance antibody stabilization.

Anti-TREM2 Antibodies

Certain aspects of the present disclosure related to anti-TREM2antibodies.

In certain embodiments, anti-TREM2 antibodies of the present disclosureare agonist antibodies that induce one or more TREM2 activities. In someembodiments, anti-TREM2 antibodies of the present disclosure are agonistantibodies that promote survival of one or more innate immune cells. Insome embodiments, anti-TREM2 antibodies of the present disclosurepromote survival of macrophages, microglial cells, M1 microglial cells,activated M1 microglial cells, M2 microglial cells, dendritic cells, M1macrophages, activated M1 macrophages, M2 macrophages, monocytes,osteoclasts, Langerhans cells of skin, and/or Kupffer cells. In someembodiments, promoting survival of one or more innate immune cellsencompasses prolonging cell survival or otherwise delaying cell death.Accordingly, in some embodiments, anti-TREM2 antibodies of the presentdisclosure prolong cell survival of one or more innate immune cells. Insome embodiments, anti-TREM2 antibodies of the present disclosure delaycell death of one or more innate immune cells. In some embodiments,promoting cell survival and/or prolonging cell survival is determined bymeasuring cell survival of one or more innate immune cells in thepresence of the anti-TREM2 antibody as compared to cell survival ofcorresponding one or more innate immune cells in the absence of theanti-TRME2 antibody. In some embodiments, delay in cell death isdetermined by measuring cell death of one or more innate immune cells inthe presence of the anti-TREM2 antibody as compared to cell death ofcorresponding one or more innate immune cells in the absence of theanti-TRME2 antibody. Any suitable methods of measuring cell survival orcell death known in the art and disclosed herein may be used (see, e.g.,Examples 30, 34, 35, 38, 52, 53, and 56). In some embodiments,anti-TREM2 antibodies of the present disclosure are agonist antibodiesthat increase IL-6 expression. In some embodiments, anti-TREM2antibodies of the present disclosure are agonist antibodies that promotesurvival of one or more innate immune cells and increase expression ofIL-6. Any suitable methods known in the art and disclosed herein formeasuring IL-6 expression in a cell may be used (see, e.g., Examples 28,38, and 68).

In certain embodiments, anti-TREM2 antibodies of the present disclosureare inert or antagonist antibodies that inhibit one or more TREM2activities. In some embodiments, anti-TREM2 antibodies of the presentdisclosure are inert or antagonist antibodies that decrease survival ofone or more innate immune cells. In some embodiments, anti-TREM2antibodies of the present disclosure decrease survival of macrophages,microglial cells, M1 microglial cells, activated M1 microglial cells, M2microglial cells, dendritic cells, M1 macrophages, activated M1macrophages, M2 macrophages, monocytes, osteoclasts, Langerhans cells ofskin, and/or Kupffer cells. In some embodiments, decreasing cellsurvival is determined by measuring cell survival of one or more innateimmune cells in the presence of the antagonist anti-TREM2 antibody ascompared to cell survival of corresponding one or more innate immunecells in the absence of the antagonist anti-TRME2 antibody. Any suitablemethods of measuring cell survival or cell death known in the art anddisclosed herein may be used (see, e.g., Examples 30, 34, 35, 38, 52,53, and 56).

In some embodiments, an isolated anti-TREM2 antibody of the presentdisclosure competes for binding of TREM2 with one or more TREM2 ligands.In some embodiments, the antibody is a human antibody, a humanizedantibody, a bispecific antibody, a multivalent antibody, or a chimericantibody. Exemplary descriptions of such antibodies are found throughoutthe present disclosure. In some embodiments, the antibody is abispecific antibody recognizing a first antigen and a second antigen.

In certain embodiments the TREM2 protein is expressed on a cell surface.In some embodiment anti-TREM2 antibodies of the present disclosuremodulate (e.g., induce or inhibit) one or more TREM2 activities. TheTREM2 activities modulated (e.g., induced or inhibited) by theanti-TREM2 antibodies may include, without limitation, DAP12phosphorylation; TREM2 phosphorylation; recruitment of Syk, ZAP70, orboth to a DAP12/TREM2 complex; PI3K activation; increased expression ofanti-inflammatory mediators (e.g. cytokines); reduced expression ofpro-inflammatory mediators; ERK phosphorylation; increased expression ofCCR7, induction of microglial cell chemotaxis toward CCL19 and CCL21expressing cells; enhancement, normalization, or both of the ability ofbone marrow-derived dendritic cells to induce antigen-specific T-cellproliferation; induction of osteoclast production, increased rate ofosteoclastogenesis, or both; increased survival and function ofmicroglial cells and/or macrophages (such as M1 macrophages and/ormicroglial cells, activated M1 macrophages and/or microglial cells,and/or M2 macrophages and/or microglial cells), dendritic cells,monocytes, osteoclasts, Langerhans cells of skin, and/or Kupffer cells;induction of apoptotic neuron clearance; reduced expression of TNF-α;SYK phosphorylation; increased expression of CD83 and/or CD86 ondendritic cells, monocytes, macrophages, and/or microglia; reducedsecretion of one or more inflammatory cytokines (such as TNF-α, IL-10,IL-6, and/or MCP-1); reduced expression of one or more inflammatoryreceptors (such as CD86); increased phagocytosis by macrophages,dendritic cells, monocytes, and/or microglia under conditions of reducedlevels of MCSF; reduced phagocytosis by macrophages, dendritic cells,monocytes, and/or microglia in the presence of normal levels of MCSF;and/or increased activity of one or more TREM2-dependent genes (e.g.,transcription factors of the nuclear factor of activated T-cells (NFAT)family of transcription factors). The anti-TREM2 antibodies of thepresent disclosure can be used to prevent, reduce risk of, or treatdementia, frontotemporal dementia, Alzheimer's disease, vasculardementia, mixed dementia, Creutzfeldt-Jakob disease, normal pressurehydrocephalus, amyotrophic lateral sclerosis, Huntington's disease,Taupathy disease, Nasu-Hakola disease, stroke, acute trauma, chronictrauma, lupus, acute and chronic colitis, wound healing, Crohn'sdisease, inflammatory bowel disease, ulcerative colitis, obesity,Malaria, essential tremor, central nervous system lupus, Behcet'sdisease, Parkinson's disease, dementia with Lewy bodies, multiple systematrophy, Shy-Drager syndrome, progressive supranuclear palsy, corticalbasal ganglionic degeneration, acute disseminated encephalomyelitis,granulomartous disorders, Sarcoidosis, diseases of aging, seizures,spinal cord injury, traumatic brain injury, age related maculardegeneration, glaucoma, retinitis pigmentosa, retinal degeneration,respiratory tract infection, sepsis, eye infection, systemic infection,lupus, arthritis, multiple sclerosis, low bone density, osteoporosis,osteogenesis, osteopetrotic disease, Paget's disease of bone, andcancer. The anti-TREM2 antibodies of the present disclosure may also beused in advanced wound care. In some embodiments, the anti-TREM2antibodies of the present disclosure are monoclonal antibodies.Anti-TREM2 antibodies of the present disclosure may be tested forinducing one or more TREM2 activities (e.g., TREM2 autophosphorylation;DAP12 phosphorylation; Syk phosphorylation; recruitment of Syk, ZAP70,or both to a DAP12/TREM2 complex; PI3K activation; increased expressionof cytokines; reduced expression of pro-inflammatory mediators; ERKphosphorylation; increased expression of CCR7; induction of microglialcell chemotaxis toward CCL19 and CCL21 expressing cells; maturation ofbone marrow-derived dendritic cells; enhancement or normalization of theability of bone marrow-derived dendritic cells to induceantigen-specific T-cell proliferation; increased ability of dendriticcells, monocytes, microglia, and/or macrophages to induce T-cellproliferation; induction of osteoclast production, increased rate ofosteoclastogenesis, or both; increased survival and function ofdendritic cells, macrophages, monocytes, osteoclasts, Langerhans cellsof skin, Kupffer cells, and/or microglia; induction of one or more typesof clearance; induction of phagocytosis of one or more of apoptoticneurons, nerve tissue debris, non-nerve tissue debris, bacteria, otherforeign bodies, disease-causing proteins, disease-causing peptides,disease-causing nucleic acid, or tumor cells; reduced secretion of oneor more inflammatory cytokines; reduced expression of one or moreinflammatory receptors; increased phagocytosis by macrophages, dendriticcells, monocytes, and/or microglial cells under conditions of reducedlevels of MCSF; reduced phagocytosis by macrophages, dendritic cells,monocytes, and/or microglial cells in the presence of normal levels ofMCSF; normalization of disrupted TREM2/DAP12-dependent gene expression;and increased activity of one or more TREM2-dependent genes) using anysuitable method known in the art and/or described herein. For example,the anti-TREM2 antibodies can be assayed in vitro for tyrosinephosphorylation of, TREM2, DAP12, Syk and/or ERK, by assaying forrecruitment of Syk and/or ZAP70 to DAP12, by assaying for PI3Kactivation, by assaying for induction of expression of cytokines (e.g.,IL-12p70, IL-6, and IL-10) or CCR7, or by assaying for reducedexpression of pro-inflammatory mediators (e.g., IL1-β and TNF) with TLRstimulation (e.g., LPS, CpG DNA, or Zymosan). Useful assays may includewestern blots (e.g., for tyrosine-phosphorylated DAP12 orthreonine/serine-phosphorylated PI3K-kinase substrates), ELISA (e.g.,for secreted interleukin or cytokine secretion), FACS (e.g., foranti-TREM2 binding to TREM2), immunocytochemistry (e.g., for e.g., fortyrosine-phosphorylated DAP12 or threonine/serine-phosphorylatedPI3K-kinase substrates), reporter-gene assays (e.g., for TLRactivation), increased survival and/or function of dendritic cells,macrophages, monocytes, osteoclasts, Langerhans cells of skin, Kupffercells, and/or microglia, increased phagocytosis of apoptotic neurons,damaged synapses, amyloid beta or fragments thereof, Tau, IAPP,alpha-synuclein, TDP-43, FUS protein, prion protein, PrPSc, huntingtin,calcitonin, superoxide dismutase, ataxin, Lewy body, atrial natriureticfactor, islet amyloid polypeptide, insulin, apolipoprotein AI, serumamyloid A, medin, prolactin, transthyretin, lysozyme, beta 2microglobulin, gelsolin, keratoepithelin, cystatin, immunoglobulin lightchain AL, S-IBM protein, Repeat-associated non-ATG (RAN) translationproducts, DiPeptide repeat (DPR) peptides, glycine-alanine (GA) repeatpeptides, glycine-proline (GP) repeat peptides, glycine-arginine (GR)repeat peptides, proline-alanine (PA) repeat peptides, andproline-arginine (PR) repeat peptides, nerve tissue debris, non-nervetissue debris, bacteria, other foreign bodies, disease-causing proteins,disease-causing peptides, disease-causing nucleic acid, or tumor cellsby macrophages, dendritic cells, Langerhans cells of skin, Kupffercells, monocytes, osteoclasts, and/or microglial cells, increasedcytoskeleton reorganization, and decreased microglial pro-inflammatoryresponses, or other assays known in the art.

In some embodiments, anti-TREM2 antibodies of the present disclosuremodulate (i.e., increase or decrease) the expression and/or secretion ofone or more inflammatory cytokines (e.g., TNF-α, IL-10, IL-6, MCP-1,IFN-a4, IFN-b, IL-1β, IL-8, CRP, TGF-beta members of the chemokineprotein families, IL-20 family members, IL-33, LIF, IFN-gamma, OSM,CNTF, TGF-beta, GM-CSF, IL-11, IL-12, IL-17, and IL-18). In someembodiments, anti-TREM2 antibodies of the present disclosure increasethe expression and/or secretion of one or more inflammatory cytokines.In some embodiments, anti-TREM2 antibodies of the present disclosuredecrease the expression and/or secretion of one or more inflammatorycytokines. In some embodiments, anti-TREM2 antibodies of the presentdisclosure modulate (i.e., increase or decrease) the expression and/orsecretion of one or more inflammatory receptors (e.g., CD86). In someembodiments, anti-TREM2 antibodies of the present disclosure increasethe expression and/or secretion of one or more inflammatory receptors.In some embodiments, anti-TREM2 antibodies of the present disclosuredecrease the expression and/or secretion of one or more inflammatoryreceptors.

In some embodiments, anti-TREM2 antibodies of the present disclosurebind to a human TREM2, or a homolog thereof, including withoutlimitation a mammalian TREM2 protein, mouse TREM2 protein (UniprotAccession No. Q99NH8), rat TREM2 protein (Uniprot Accession No. D3ZZ89),Rhesus monkey TREM2 protein (Uniprot Accession No. F6QVF2), bovine TREM2protein (Uniprot Accession No. Q05B59), equine TREM2 protein (UniprotAccession No. F7D6L0), pig TREM2 protein (Uniprot Accession No. H2EZZ3),and dog TREM2 protein (Uniprot Accession No. E2RP46). In someembodiments, anti-TREM2 antibodies of the present disclosurespecifically bind to human TREM2. In some embodiments, anti-TREM2antibodies of the present disclosure specifically bind to mouse TREM2.In some embodiments, anti-TREM2 antibodies of the present disclosurespecifically bind to both human TREM2 and mouse TREM2. In someembodiments, anti-TREM2 antibodies of the present disclosure modulate(e.g., induce or inhibit) at least one TREM2 activity. In someembodiments, the at least one TREM2 activity is DAP12 phosphorylation,TREM2 phosphorylation, PI3K activation, increased expression of one ormore anti-inflammatory mediators (e.g., cytokines), reduced expressionof one or more pro-inflammatory mediators, increased survival and/orfunction of microglial cells, dendritic cells, macrophages, monocytes,osteoclasts, Langerhans cells of skin, and/or Kupffer cells, reducedexpression of TNF-α, SYK phosphorylation, increased expression of CD83and/or CD86 on dendritic cells, macrophages, monocytes, and/ormacrophages, reduced secretion of one or more inflammatory cytokines,reduced expression of one or more inflammatory receptors, increasedphagocytosis by macrophages, dendritic cells, monocytes, and/ormicroglia under conditions of reduced levels of MCSF, reducedphagocytosis by macrophages, dendritic cells, monocytes, and/ormicroglia in the presence of normal levels of MCSF, and/or increasedactivity of one or more TREM2-dependent genes (e.g., transcriptionfactors of the nuclear factor of activated T-cells (NFAT) family oftranscription factors).

In some embodiments, anti-TREM2 antibodies of the present disclosurebind to a TREM2 protein of the present disclosure and/or naturallyoccurring variants. In certain preferred embodiments, the anti-TREM2antibodies bind to human TREM2.

In some embodiments, anti-TREM2 antibodies of the present disclosure areagonist antibodies, or antagonist antibodies that bind to a TREM2protein of the present disclosure expressed on the surface of a cell andmodulate (e.g., induce or inhibit) at least one TREM2 activity of thepresent disclosure after binding to the surface-expressed TREM2 protein.In some embodiments, anti-TREM2 antibodies of the present disclosure areinert antibodies.

In certain embodiments, anti-TREM2 antibodies of the present disclosurebind to one or more amino acids within amino acid residues 29-112 ofhuman TREM 2 (SEQ ID NO: 1), or within amino acid residues on a TREM2protein corresponding to amino acid residues 29-112 of SEQ ID NO: 1. Insome embodiments, anti-TREM2 antibodies of the present disclosure bindto one or more amino acids within amino acid residues 29-41 of humanTREM 2 (SEQ ID NO: 1), or within amino acid residues on a TREM2 proteincorresponding to amino acid residues 29-41 of SEQ ID NO: 1. In someembodiments, anti-TREM2 antibodies of the present disclosure bind to oneor more amino acids within amino acid residues 47-69 of human TREM 2(SEQ ID NO: 1), or within amino acid residues on a TREM2 proteincorresponding to amino acid residues 47-69 of SEQ ID NO: 1. In someembodiments, anti-TREM2 antibodies of the present disclosure bind to oneor more amino acids within amino acid residues 76-86 of human TREM 2(SEQ ID NO: 1), or within amino acid residues on a TREM2 proteincorresponding to amino acid residues 76-86 of SEQ ID NO: 1. In someembodiments, anti-TREM2 antibodies of the present disclosure bind to oneor more amino acids within amino acid residues 91-100 of human TREM 2(SEQ ID NO: 1), or within amino acid residues on a TREM2 proteincorresponding to amino acid residues 91-100 of SEQ ID NO: 1. In someembodiments, anti-TREM2 antibodies of the present disclosure bind to oneor more amino acids within amino acid residues 99-115 of human TREM 2(SEQ ID NO: 1), or within amino acid residues on a TREM2 proteincorresponding to amino acid residues 99-115 of SEQ ID NO: 1. In someembodiments, anti-TREM2 antibodies of the present disclosure bind to oneor more amino acids within amino acid residues 104-112 of human TREM 2(SEQ ID NO: 1), or within amino acid residues on a TREM2 proteincorresponding to amino acid residues 104-112 of SEQ ID NO: 1. In someembodiments, anti-TREM2 antibodies of the present disclosure bind to oneor more amino acids within amino acid residues 114-118 of human TREM 2(SEQ ID NO: 1), or within amino acid residues on a TREM2 proteincorresponding to amino acid residues 114-118 of SEQ ID NO: 1. In someembodiments, anti-TREM2 antibodies of the present disclosure bind to oneor more amino acids within amino acid residues 130-171 of human TREM 2(SEQ ID NO: 1), or within amino acid residues on a TREM2 proteincorresponding to amino acid residues 130-171 of SEQ ID NO: 1. In someembodiments, anti-TREM2 antibodies of the present disclosure bind to oneor more amino acids within amino acid residues 139-153 of human TREM 2(SEQ ID NO: 1), or within amino acid residues on a TREM2 proteincorresponding to amino acid residues 139-153 of SEQ ID NO: 1. In someembodiments, anti-TREM2 antibodies of the present disclosure bind to oneor more amino acids within amino acid residues 139-146 of human TREM 2(SEQ ID NO: 1), or within amino acid residues on a TREM2 proteincorresponding to amino acid residues 139-146 of SEQ ID NO: 1. In someembodiments, anti-TREM2 antibodies of the present disclosure bind to oneor more amino acids within amino acid residues 130-144 of human TREM 2(SEQ ID NO: 1), or within amino acid residues on a TREM2 proteincorresponding to amino acid residues 130-144 of SEQ ID NO: 1. In someembodiments, anti-TREM2 antibodies of the present disclosure bind to oneor more amino acids within amino acid residues 158-171 of human TREM 2(SEQ ID NO: 1), or within amino acid residues on a TREM2 proteincorresponding to amino acid residues 158-171 of SEQ ID NO: 1.

In some embodiments, anti-TREM2 antibodies of the present disclosurebind to one or more amino acids within amino acid residues 43-50 ofhuman TREM 2 (SEQ ID NO: 1), or within amino acid residues on a TREM2protein corresponding to amino acid residues 43-50 of SEQ ID NO: 1. Insome embodiments, anti-TREM2 antibodies of the present disclosure bindto one or more amino acids within amino acid residues 49-57 of humanTREM 2 (SEQ ID NO: 1), or within amino acid residues on a TREM2 proteincorresponding to amino acid residues 49-57 of SEQ ID NO: 1. In someembodiments, anti-TREM2 antibodies of the present disclosure bind to oneor more amino acids within amino acid residues 139-146 of human TREM 2(SEQ ID NO: 1), or within amino acid residues on a TREM2 proteincorresponding to amino acid residues 139-146 of SEQ ID NO: 1. In someembodiments, anti-TREM2 antibodies of the present disclosure bind to oneor more amino acids within amino acid residues 140-153 of human TREM 2(SEQ ID NO: 1), or within amino acid residues on a TREM2 proteincorresponding to amino acid residues 140-153 of SEQ ID NO: 1.

TREM2 proteins of the present disclosure include a complementarydetermining region 1 (CDR1) located at amino acid residues correspondingto amino acid residues 40-44 of human TREM2 (SEQ ID NO: 1); acomplementary determining region 2 (CDR2) located at amino acid residuescorresponding to amino acid residues 67-76 of human TREM2 (SEQ ID NO:1); and a complementary determining region 3 (CDR3) located at aminoacid residues corresponding to amino acid residues 114-118 of humanTREM2 (SEQ ID NO: 1). Accordingly, in some embodiments, anti-TREM2antibodies of the present disclosure bind to one or more amino acidswithin amino acid residues 40-44 of human TREM 2 (SEQ ID NO: 1), orwithin amino acid residues on a TREM2 protein corresponding to aminoacid residues 40-44 of SEQ ID NO: 1. In some embodiments, anti-TREM2antibodies of the present disclosure bind to one or more amino acidswithin amino acid residues 67-76 of human TREM 2 (SEQ ID NO: 1), orwithin amino acid residues on a TREM2 protein corresponding to aminoacid residues 67-76 of SEQ ID NO: 1. In some embodiments, anti-TREM2antibodies of the present disclosure bind to one or more amino acidswithin amino acid residues 114-118 of human TREM 2 (SEQ ID NO: 1), orwithin amino acid residues on a TREM2 protein corresponding to aminoacid residues 114-118 of SEQ ID NO: 1.

In other embodiments, anti-TREM2 antibodies of the present disclosurebind to an epitope that includes amino acid residue Arg47 or Asp87 ofhuman TREM 2 (SEQ ID NO: 1). In some embodiments, anti-TREM2 antibodiesof the present disclosure bind to an epitope that includes amino acidresidues 40-44 of human TREM 2 (SEQ ID NO: 1). In some embodiments,anti-TREM2 antibodies of the present disclosure bind to an epitope thatincludes amino acid residues 67-76 of human TREM 2 (SEQ ID NO: 1). Insome embodiments, anti-TREM2 antibodies of the present disclosure bindto an epitope that includes amino acid residues 114-118 of human TREM 2(SEQ ID NO: 1).

In some embodiments, anti-TREM2 antibodies of the present disclosurecompetitively inhibit binding of at least one antibody selected from anyof the antibodies listed in Table land/or Table 8. In some embodiments,anti-TREM2 antibodies of the present disclosure competitively inhibitbinding of at least one antibody selected from Ab1, Ab2, Ab3, Ab4, Ab5,Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16, Ab17,Ab18, Ab19, Ab20, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29,Ab30, Ab31, Ab32, Ab33, Ab34, Ab35, Ab36, Ab37, Ab38, Ab39, Ab40, Ab41,Ab42, Ab43, Ab44, Ab45, Ab46, Ab47, Ab48, Ab49, Ab50, Ab51, Ab52, Ab53,Ab54, Ab55, Ab56, Ab57, Ab58, Ab59, Ab60, Ab61, Ab62, Ab63, Ab64, Ab65,Ab66, Ab67, Ab68, Ab69, Ab70, Ab71, Ab72, Ab73, Ab74, Ab75, Ab76, Ab77,Ab78, Ab79, Ab80, Ab81, Ab82, Ab83, Ab84, Ab85, Ab86, and Ab87. In someembodiments, anti-TREM2 antibodies of the present disclosurecompetitively inhibit binding of at least one of the followinganti-TREM2 antibodies: Ab1, Ab9, Ab14, Ab22, Ab45, and Ab65. In someembodiments, anti-TREM2 antibodies of the present disclosure bind to anepitope of human TREM2 that is the same as or overlaps with the TREM2epitope bound by at least one antibody selected from any of theantibodies listed in Table land/or Table 8. In some embodiments,anti-TREM2 antibodies of the present disclosure bind to an epitope ofhuman TREM2 that is the same as or overlaps with the TREM2 epitope boundby at least one antibody selected from Ab1, Ab2, Ab3, Ab4, Ab5, Ab6,Ab7, Ab8, Ab9, Ab10, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18,Ab19, Ab20, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30,Ab31, Ab32, Ab33, Ab34, Ab35, Ab36, Ab37, Ab38, Ab39, Ab40, Ab41, Ab42,Ab43, Ab44, Ab45, Ab46, Ab47, Ab48, Ab49, Ab50, Ab51, Ab52, Ab53, Ab54,Ab55, Ab56, Ab57, Ab58, Ab59, Ab60, Ab61, Ab62, Ab63, Ab64, Ab65, Ab66,Ab67, Ab68, Ab69, Ab70, Ab71, Ab72, Ab73, Ab74, Ab75, Ab76, Ab77, Ab78,Ab79, Ab80, Ab81, Ab82, Ab83, Ab84, Ab85, Ab86, and Ab87. In someembodiments, anti-TREM2 antibodies of the present disclosure bind to anepitope of human TREM2 that is the same as or overlaps with the TREM2epitope bound by at least one of the following anti-TREM2 antibodies:Ab1, Ab9, Ab14, Ab22, Ab45, and Ab65. In some embodiments, anti-TREM2antibodies of the present disclosure bind essentially the same TREM2epitope bound by at least one antibody selected from any of theantibodies listed in Table land/or Table 8. In some embodiments,anti-TREM2 antibodies of the present disclosure bind essentially thesame TREM2 epitope bound by at least one antibody selected from Ab1,Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab12, Ab13, Ab14,Ab15, Ab16, Ab17, Ab18, Ab19, Ab20, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26,Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, Ab34, Ab35, Ab36, Ab37, Ab38,Ab39, Ab40, Ab41, Ab42, Ab43, Ab44, Ab45, Ab46, Ab47, Ab48, Ab49, Ab50,Ab51, Ab52, Ab53, Ab54, Ab55, Ab56, Ab57, Ab58, Ab59, Ab60, Ab61, Ab62,Ab63, Ab64, Ab65, Ab66, Ab67, Ab68, Ab69, Ab70, Ab71, Ab72, Ab73, Ab74,Ab75, Ab76, Ab77, Ab78, Ab79, Ab80, Ab81, Ab82, Ab83, Ab84, Ab85, Ab86,and Ab87. In some embodiments, anti-TREM2 antibodies of the presentdisclosure bind essentially the same TREM2 epitope bound by at least oneof the following anti-TREM2 antibodies: Ab1, Ab9, Ab14, Ab22, Ab45, andAb65. Detailed exemplary methods for mapping an epitope to which anantibody binds are provided in Morris (1996) “Epitope MappingProtocols,” in Methods in Molecular Biology vol. 66 (Humana Press,Totowa, N.J.).

In an exemplary competition assay, immobilized TREM2 or cells expressingTEM2 on the cell surface are incubated in a solution comprising a firstlabeled antibody that binds to TREM2 (e.g., human or non-human primate)and a second unlabeled antibody that is being tested for its ability tocompete with the first antibody for binding to TREM2. The secondantibody may be present in a hybridoma supernatant. As a control,immobilized TREM2 or cells expressing TREM2 is incubated in a solutioncomprising the first labeled antibody but not the second unlabeledantibody. After incubation under conditions permissive for binding ofthe first antibody to TREM2, excess unbound antibody is removed, and theamount of label associated with immobilized TREM2 or cells expressingTREM2 is measured. If the amount of label associated with immobilizedTREM2 or cells expressing TREM2 is substantially reduced in the testsample relative to the control sample, then that indicates that thesecond antibody is competing with the first antibody for binding toTREM2. See, Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

In some embodiments, anti-TREM2 antibodies of the present disclosurecomprise (a) a heavy chain variable region comprising at least one, two,or three HVRs selected from HVR-H1, HVR-H2, and HVR-H3 of any one of theantibodies listed in Table land/or Table 8 or selected from Ab1, Ab2,Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab12, Ab13, Ab14, Ab15,Ab16, Ab17, Ab18, Ab19, Ab20, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27,Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, Ab34, Ab35, Ab36, Ab37, Ab38, Ab39,Ab40, Ab41, Ab42, Ab43, Ab44, Ab45, Ab46, Ab47, Ab48, Ab49, Ab50, Ab51,Ab52, Ab53, Ab54, Ab55, Ab56, Ab57, Ab58, Ab59, Ab60, Ab61, Ab62, Ab63,Ab64, Ab65, Ab66, Ab67, Ab68, Ab69, Ab70, Ab71, Ab72, Ab73, Ab74, Ab75,Ab76, Ab77, Ab78, Ab79, Ab80, Ab81, Ab82, Ab83, Ab84, Ab85, Ab86, andAb87; and/or (b) a light chain variable region comprising at least one,two, or three HVRs selected from HVR-L1, HVR-L2, and HVR-L3 of any oneof the antibodies listed in Table land/or Table 8 or selected from Ab1,Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab12, Ab13, Ab14,Ab15, Ab16, Ab17, Ab18, Ab19, Ab20, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26,Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, Ab34, Ab35, Ab36, Ab37, Ab38,Ab39, Ab40, Ab41, Ab42, Ab43, Ab44, Ab45, Ab46, Ab47, Ab48, Ab49, Ab50,Ab51, Ab52, Ab53, Ab54, Ab55, Ab56, Ab57, Ab58, Ab59, Ab60, Ab61, Ab62,Ab63, Ab64, Ab65, Ab66, Ab67, Ab68, Ab69, Ab70, Ab71, Ab72, Ab73, Ab74,Ab75, Ab76, Ab77, Ab78, Ab79, Ab80, Ab81, Ab82, Ab83, Ab84, Ab85, Ab86,and Ab87. In some embodiments, the HVR-H1, HVR-H2, HVR-H3, HVR-L1,HVR-L2, and HVR-L3 comprise Kabat CDR, Chothia CDR, or Contact CDRsequences as shown in Table land/or Table 8 or from an antibody selectedfrom Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab12,Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19, Ab20, Ab21, Ab22, Ab23, Ab24,Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, Ab34, Ab35, Ab36,Ab37, Ab38, Ab39, Ab40, Ab41, Ab42, Ab43, Ab44, Ab45, Ab46, Ab47, Ab48,Ab49, Ab50, Ab51, Ab52, Ab53, Ab54, Ab55, Ab56, Ab57, Ab58, Ab59, Ab60,Ab61, Ab62, Ab63, Ab64, Ab65, Ab66, Ab67, Ab68, Ab69, Ab70, Ab71, Ab72,Ab73, Ab74, Ab75, Ab76, Ab77, Ab78, Ab79, Ab80, Ab81, Ab82, Ab83, Ab84,Ab85, Ab86, and Ab87.

In some embodiments, anti-TREM2 antibodies of the present disclosurecomprise at least one, two, three, four, five, or six HVRs selected from(i) HVR-H1 comprising the amino acid sequence of any of the HVR-H1sequences listed in Table land/or Table 8 or from an antibody selectedfrom Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab12,Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19, Ab20, Ab21, Ab22, Ab23, Ab24,Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, Ab34, Ab35, Ab36,Ab37, Ab38, Ab39, Ab40, Ab41, Ab42, Ab43, Ab44, Ab45, Ab46, Ab47, Ab48,Ab49, Ab50, Ab51, Ab52, Ab53, Ab54, Ab55, Ab56, Ab57, Ab58, Ab59, Ab60,Ab61, Ab62, Ab63, Ab64, Ab65, Ab66, Ab67, Ab68, Ab69, Ab70, Ab71, Ab72,Ab73, Ab74, Ab75, Ab76, Ab77, Ab78, Ab79, Ab80, Ab81, Ab82, Ab83, Ab84,Ab85, Ab86, and Ab87; (ii) HVR-H2 comprising the amino acid sequence ofany of the HVR-H2 sequences listed in Table land/or Table 8 or from anantibody selected from Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9,Ab10, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19, Ab20, Ab21,Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33,Ab34, Ab35, Ab36, Ab37, Ab38, Ab39, Ab40, Ab41, Ab42, Ab43, Ab44, Ab45,Ab46, Ab47, Ab48, Ab49, Ab50, Ab51, Ab52, Ab53, Ab54, Ab55, Ab56, Ab57,Ab58, Ab59, Ab60, Ab61, Ab62, Ab63, Ab64, Ab65, Ab66, Ab67, Ab68, Ab69,Ab70, Ab71, Ab72, Ab73, Ab74, Ab75, Ab76, Ab77, Ab78, Ab79, Ab80, Ab81,Ab82, Ab83, Ab84, Ab85, Ab86, and Ab87; (iii) HVR-H3 comprising theamino acid sequence of any of the HVR-H3 sequences listed in Tableland/or Table 8 or from an antibody selected from Ab1, Ab2, Ab3, Ab4,Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16, Ab17,Ab18, Ab19, Ab20, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29,Ab30, Ab31, Ab32, Ab33, Ab34, Ab35, Ab36, Ab37, Ab38, Ab39, Ab40, Ab41,Ab42, Ab43, Ab44, Ab45, Ab46, Ab47, Ab48, Ab49, Ab50, Ab51, Ab52, Ab53,Ab54, Ab55, Ab56, Ab57, Ab58, Ab59, Ab60, Ab61, Ab62, Ab63, Ab64, Ab65,Ab66, Ab67, Ab68, Ab69, Ab70, Ab71, Ab72, Ab73, Ab74, Ab75, Ab76, Ab77,Ab78, Ab79, Ab80, Ab81, Ab82, Ab83, Ab84, Ab85, Ab86, and Ab87; (iv)HVR-L1 comprising the amino acid sequence of any of the HVR-L1 sequenceslisted in Table land/or Table 8 or from an antibody selected from Ab1,Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab12, Ab13, Ab14,Ab15, Ab16, Ab17, Ab18, Ab19, Ab20, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26,Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, Ab34, Ab35, Ab36, Ab37, Ab38,Ab39, Ab40, Ab41, Ab42, Ab43, Ab44, Ab45, Ab46, Ab47, Ab48, Ab49, Ab50,Ab51, Ab52, Ab53, Ab54, Ab55, Ab56, Ab57, Ab58, Ab59, Ab60, Ab61, Ab62,Ab63, Ab64, Ab65, Ab66, Ab67, Ab68, Ab69, Ab70, Ab71, Ab72, Ab73, Ab74,Ab75, Ab76, Ab77, Ab78, Ab79, Ab80, Ab81, Ab82, Ab83, Ab84, Ab85, Ab86,and Ab87; (v) HVR-L2 comprising the amino acid sequence of any of theHVR-L2 sequences listed in Table land/or Table 8 or from an antibodyselected from Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11,Ab12, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19, Ab20, Ab21, Ab22, Ab23,Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, Ab34, Ab35,Ab36, Ab37, Ab38, Ab39, Ab40, Ab41, Ab42, Ab43, Ab44, Ab45, Ab46, Ab47,Ab48, Ab49, Ab50, Ab51, Ab52, Ab53, Ab54, Ab55, Ab56, Ab57, Ab58, Ab59,Ab60, Ab61, Ab62, Ab63, Ab64, Ab65, Ab66, Ab67, Ab68, Ab69, Ab70, Ab71,Ab72, Ab73, Ab74, Ab75, Ab76, Ab77, Ab78, Ab79, Ab80, Ab81, Ab82, Ab83,Ab84, Ab85, Ab86, and Ab87; and (vi) HVR-L3 comprising the amino acidsequence of any of the HVR-L3 sequences listed in Table land/or Table 8or from an antibody selected from Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7,Ab8, Ab9, Ab10, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19,Ab20, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31,Ab32, Ab33, Ab34, Ab35, Ab36, Ab37, Ab38, Ab39, Ab40, Ab41, Ab42, Ab43,Ab44, Ab45, Ab46, Ab47, Ab48, Ab49, Ab50, Ab51, Ab52, Ab53, Ab54, Ab55,Ab56, Ab57, Ab58, Ab59, Ab60, Ab61, Ab62, Ab63, Ab64, Ab65, Ab66, Ab67,Ab68, Ab69, Ab70, Ab71, Ab72, Ab73, Ab74, Ab75, Ab76, Ab77, Ab78, Ab79,Ab80, Ab81, Ab82, Ab83, Ab84, Ab85, Ab86, and Ab87.

In some embodiments, anti-TREM2 antibodies of the present disclosurecomprise a heavy chain variable domain and a light chain variabledomain, wherein the heavy chain variable domain comprises one or moreof: (a) an HVR-H1 comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs:3-24, or an amino acid sequence with atleast about 95% homology to an amino acid sequence selected from thegroup consisting of SEQ ID NOs:3-24; (b) an HVR-H2 comprising an aminoacid sequence selected from the group consisting of SEQ ID NOs:25-49, oran amino acid sequence with at least about 95% homology to an amino acidsequence selected from the group consisting of SEQ ID NOs: 25-49; and(c) an HVR-H3 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs:50-119, or an amino acid sequence with at leastabout 95% homology to an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 50-119; and/or wherein the light chainvariable domain comprises one or more of: (a) an HVR-L1 comprising anamino acid sequence selected from the group consisting of SEQ IDNOs:120-137, or an amino acid sequence with at least about 95% homologyto an amino acid sequence selected from the group consisting of SEQ IDNOs:120-137; (b) an HVR-L2 comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NOs:138-152, or an amino acidsequence with at least about 95% homology to an amino acid sequenceselected from the group consisting of SEQ ID NOs:138-152; and (c) anHVR-L3 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs:153-236 or an amino acid sequence with at leastabout 95% homology to an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 138-152.

In some embodiments, anti-TREM2 antibodies of the present disclosurecomprise a heavy chain variable region of any one of the antibodieslisted in Table land/or Table 8 or selected from Ab1, Ab2, Ab3, Ab4,Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16, Ab17,Ab18, Ab19, Ab20, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29,Ab30, Ab31, Ab32, Ab33, Ab34, Ab35, Ab36, Ab37, Ab38, Ab39, Ab40, Ab41,Ab42, Ab43, Ab44, Ab45, Ab46, Ab47, Ab48, Ab49, Ab50, Ab51, Ab52, Ab53,Ab54, Ab55, Ab56, Ab57, Ab58, Ab59, Ab60, Ab61, Ab62, Ab63, Ab64, Ab65,Ab66, Ab67, Ab68, Ab69, Ab70, Ab71, Ab72, Ab73, Ab74, Ab75, Ab76, Ab77,Ab78, Ab79, Ab80, Ab81, Ab82, Ab83, Ab84, Ab85, Ab86, and Ab87; and/or alight chain variable region of any one of the antibodies listed in Tableland/or Table 8 or selected from Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8,Ab9, Ab10, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19, Ab20,Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32,Ab33, Ab34, Ab35, Ab36, Ab37, Ab38, Ab39, Ab40, Ab41, Ab42, Ab43, Ab44,Ab45, Ab46, Ab47, Ab48, Ab49, Ab50, Ab51, Ab52, Ab53, Ab54, Ab55, Ab56,Ab57, Ab58, Ab59, Ab60, Ab61, Ab62, Ab63, Ab64, Ab65, Ab66, Ab67, Ab68,Ab69, Ab70, Ab71, Ab72, Ab73, Ab74, Ab75, Ab76, Ab77, Ab78, Ab79, Ab80,Ab81, Ab82, Ab83, Ab84, Ab85, Ab86, and Ab87.

Any of the antibodies of the present disclosure may be produced by acell line. In some embodiments, the cell line may be a yeast cell line.In other embodiments, the cell line may be a mammalian cell line. Incertain embodiments, the cell line may be a hybridoma cell line. Anycell line known in the art suitable for antibody production may be usedto produce an antibody of the present disclosure. Exemplary cell linesfor antibody production are described throughout the present disclosure.

In some embodiments, the anti-TREM2 antibody is an anti-TREM2 monoclonalantibody selected from Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9,Ab10, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19, Ab20, Ab21,Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33,Ab34, Ab35, Ab36, Ab37, Ab38, Ab39, Ab40, Ab41, Ab42, Ab43, Ab44, Ab45,Ab46, Ab47, Ab48, Ab49, Ab50, Ab51, Ab52, Ab53, Ab54, Ab55, Ab56, Ab57,Ab58, Ab59, Ab60, Ab61, Ab62, Ab63, Ab64, Ab65, Ab66, Ab67, Ab68, Ab69,Ab70, Ab71, Ab72, Ab73, Ab74, Ab75, Ab76, Ab77, Ab78, Ab79, Ab80, Ab81,Ab82, Ab83, Ab84, Ab85, Ab86, and Ab87. In certain embodiments, theanti-TREM2 antibody is an agonist antibody. In other embodiments, theanti-TREM2 antibody is an antagonist antibody.

In some embodiments, the anti-TREM2 antibody is anti-TREM2 monoclonalantibody Ab1. In some embodiments, the anti-TREM2 antibody is anisolated antibody which binds essentially the same TREM2 epitope as Ab1.In some embodiments, the anti-TREM2 antibody is an isolated antibodycomprising the HVR-H1, HVR-H2, and HVR-H3 of the heavy chain variabledomains of monoclonal antibody Ab1. In some embodiments, the anti-TREM2antibody is an isolated antibody comprising the HVR-L1, HVR-L2, andHVR-L3 of the light chain variable domains of monoclonal antibody Ab1.In some embodiments, the anti-TREM2 antibody is an isolated antibodycomprising the HVR-H1, HVR-H2, and HVR-H3 of the heavy chain variabledomains and the HVR-L1, HVR-L2, and HVR-L3 of the light chain variabledomains of monoclonal antibody Ab1. In certain embodiments, theanti-TREM2 antibody is an agonist antibody. In other embodiments, theanti-TREM2 antibody is an antagonist antibody. In some embodiments,anti-TREM2 antibodies of the present disclosure comprise at least one,two, three, four, five, or six HVRs selected from (i) HVR-H1 comprisingthe amino acid sequence of SEQ ID NO:3, or an amino acid sequence withat least about 95% homology to the amino acid sequence of SEQ ID NO:3;(ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 25, or anamino acid sequence with at least about 95% homology to the amino acidsequence of SEQ ID NO:25; (iii) HVR-H3 comprising the amino acidsequence of SEQ ID NO:50, or an amino acid sequence with at least about95% homology to the amino acid sequence of SEQ ID NO:50; (iv) HVR-L1comprising the amino acid sequence of SEQ ID NO: 120 or an amino acidsequence with at least about 95% homology to the amino acid sequence ofSEQ ID NO: 120; (v) HVR-L2 comprising the amino acid sequence of SEQ IDNO: 138 or an amino acid sequence with at least about 95% homology tothe amino acid sequence of SEQ ID NO: 138; and (vi) HVR-L3 comprisingthe amino acid sequence of SEQ ID NO: 153, or an amino acid sequencewith at least about 95% homology to the amino acid sequence of SEQ IDNO: 153.

In some embodiments, the anti-TREM2 antibody is anti-TREM2 monoclonalantibody Ab9. In some embodiments, the anti-TREM2 antibody is anisolated antibody which binds essentially the same TREM2 epitope as Ab9.In some embodiments, the anti-TREM2 antibody is an isolated antibodycomprising the HVR-H1, HVR-H2, and HVR-H3 of the heavy chain variabledomains of monoclonal antibody Ab9. In some embodiments, the anti-TREM2antibody is an isolated antibody comprising the HVR-L1, HVR-L2, andHVR-L3 of the light chain variable domains of monoclonal antibody Ab9.In some embodiments, the anti-TREM2 antibody is an isolated antibodycomprising the HVR-H1, HVR-H2, and HVR-H3 of the heavy chain variabledomains and the HVR-L1, HVR-L2, and HVR-L3 of the light chain variabledomains of monoclonal antibody Ab9. In certain embodiments, theanti-TREM2 antibody is an agonist antibody. In other embodiments, theanti-TREM2 antibody is an antagonist antibody. In some embodiments,anti-TREM2 antibodies of the present disclosure comprise at least one,two, three, four, five, or six HVRs selected from (i) HVR-H1 comprisingthe amino acid sequence of SEQ ID NO:9, or an amino acid sequence withat least about 95% homology to the amino acid sequence of SEQ ID NO:9;(ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 33, or anamino acid sequence with at least about 95% homology to the amino acidsequence of SEQ ID NO:33; (iii) HVR-H3 comprising the amino acidsequence of SEQ ID NO:58, or an amino acid sequence with at least about95% homology to the amino acid sequence of SEQ ID NO:58; (iv) HVR-L1comprising the amino acid sequence of SEQ ID NO: 124 or an amino acidsequence with at least about 95% homology to the amino acid sequence ofSEQ ID NO:124; (v) HVR-L2 comprising the amino acid sequence of SEQ IDNO: 144 or an amino acid sequence with at least about 95% homology tothe amino acid sequence of SEQ ID NO: 144; and (vi) HVR-L3 comprisingthe amino acid sequence of SEQ ID NO: 161, or an amino acid sequencewith at least about 95% homology to the amino acid sequence of SEQ IDNO: 161.

In some embodiments, the anti-TREM2 antibody is anti-TREM2 monoclonalantibody Ab14. In some embodiments, the anti-TREM2 antibody is anisolated antibody which binds essentially the same TREM2 epitope asAb14. In some embodiments, the anti-TREM2 antibody is an isolatedantibody comprising the HVR-H1, HVR-H2, and HVR-H3 of the heavy chainvariable domains of monoclonal antibody Ab14. In some embodiments, theanti-TREM2 antibody is an isolated antibody comprising the HVR-L1,HVR-L2, and HVR-L3 of the light chain variable domains of monoclonalantibody Ab14. In some embodiments, the anti-TREM2 antibody is anisolated antibody comprising the HVR-H1, HVR-H2, and HVR-H3 of the heavychain variable domains and the HVR-L1, HVR-L2, and HVR-L3 of the lightchain variable domains of monoclonal antibody Ab14. In certainembodiments, the anti-TREM2 antibody is an agonist antibody. In otherembodiments, the anti-TREM2 antibody is an antagonist antibody. In someembodiments, anti-TREM2 antibodies of the present disclosure comprise atleast one, two, three, four, five, or six HVRs selected from (i) HVR-H1comprising the amino acid sequence of SEQ ID NO: 13, or an amino acidsequence with at least about 95% homology to the amino acid sequence ofSEQ ID NO: 13; (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO: 36, or an amino acid sequence with at least about 95% homology tothe amino acid sequence of SEQ ID NO:36; (iii) HVR-H3 comprising theamino acid sequence of SEQ ID NO:63, or an amino acid sequence with atleast about 95% homology to the amino acid sequence of SEQ ID NO:63;(iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122 or anamino acid sequence with at least about 95% homology to the amino acidsequence of SEQ ID NO:122; (v) HVR-L2 comprising the amino acid sequenceof SEQ ID NO: 146 or an amino acid sequence with at least about 95%homology to the amino acid sequence of SEQ ID NO: 146; and (vi) HVR-L3comprising the amino acid sequence of SEQ ID NO: 166, or an amino acidsequence with at least about 95% homology to the amino acid sequence ofSEQ ID NO: 166.

In some embodiments, the anti-TREM2 antibody is anti-TREM2 monoclonalantibody Ab22. In some embodiments, the anti-TREM2 antibody is anisolated antibody which binds essentially the same TREM2 epitope asAb22. In some embodiments, the anti-TREM2 antibody is an isolatedantibody comprising the HVR-H1, HVR-H2, and HVR-H3 of the heavy chainvariable domains of monoclonal antibody Ab22. In some embodiments, theanti-TREM2 antibody is an isolated antibody comprising the HVR-L1,HVR-L2, and HVR-L3 of the light chain variable domains of monoclonalantibody Ab22. In some embodiments, the anti-TREM2 antibody is anisolated antibody comprising the HVR-H1, HVR-H2, and HVR-H3 of the heavychain variable domains and the HVR-L1, HVR-L2, and HVR-L3 of the lightchain variable domains of monoclonal antibody Ab22. In certainembodiments, the anti-TREM2 antibody is an agonist antibody. In otherembodiments, the anti-TREM2 antibody is an antagonist antibody. In someembodiments, anti-TREM2 antibodies of the present disclosure comprise atleast one, two, three, four, five, or six HVRs selected from (i) HVR-H1comprising the amino acid sequence of SEQ ID NO: 11, or an amino acidsequence with at least about 95% homology to the amino acid sequence ofSEQ ID NO:11; (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO: 34, or an amino acid sequence with at least about 95% homology tothe amino acid sequence of SEQ ID NO:34; (iii) HVR-H3 comprising theamino acid sequence of SEQ ID NO:60, or an amino acid sequence with atleast about 95% homology to the amino acid sequence of SEQ ID NO:60;(iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 123 or anamino acid sequence with at least about 95% homology to the amino acidsequence of SEQ ID NO: 123; (v) HVR-L2 comprising the amino acidsequence of SEQ ID NO: 141 or an amino acid sequence with at least about95% homology to the amino acid sequence of SEQ ID NO: 141; and (vi)HVR-L3 comprising the amino acid sequence of SEQ ID NO: 173, or an aminoacid sequence with at least about 95% homology to the amino acidsequence of SEQ ID NO: 173.

In some embodiments, the anti-TREM2 antibody is anti-TREM2 monoclonalantibody Ab45. In some embodiments, the anti-TREM2 antibody is anisolated antibody which binds essentially the same TREM2 epitope asAb45. In some embodiments, the anti-TREM2 antibody is an isolatedantibody comprising the HVR-H1, HVR-H2, and HVR-H3 of the heavy chainvariable domains of monoclonal antibody Ab45. In some embodiments, theanti-TREM2 antibody is an isolated antibody comprising the HVR-L1,HVR-L2, and HVR-L3 of the light chain variable domains of monoclonalantibody Ab45. In some embodiments, the anti-TREM2 antibody is anisolated antibody comprising the HVR-H1, HVR-H2, and HVR-H3 of the heavychain variable domains and the HVR-L1, HVR-L2, and HVR-L3 of the lightchain variable domains of monoclonal antibody Ab45. In certainembodiments, the anti-TREM2 antibody is an agonist antibody. In otherembodiments, the anti-TREM2 antibody is an antagonist antibody. In someembodiments, anti-TREM2 antibodies of the present disclosure comprise atleast one, two, three, four, five, or six HVRs selected from (i) HVR-H1comprising the amino acid sequence of SEQ ID NO:7, or an amino acidsequence with at least about 95% homology to the amino acid sequence ofSEQ ID NO:7; (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO: 29, or an amino acid sequence with at least about 95% homology tothe amino acid sequence of SEQ ID NO:29; (iii) HVR-H3 comprising theamino acid sequence of SEQ ID NO:87, or an amino acid sequence with atleast about 95% homology to the amino acid sequence of SEQ ID NO:87;(iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 120 or anamino acid sequence with at least about 95% homology to the amino acidsequence of SEQ ID NO: 120; (v) HVR-L2 comprising the amino acidsequence of SEQ ID NO:138 or an amino acid sequence with at least about95% homology to the amino acid sequence of SEQ ID NO: 138; and (vi)HVR-L3 comprising the amino acid sequence of SEQ ID NO: 196, or an aminoacid sequence with at least about 95% homology to the amino acidsequence of SEQ ID NO: 196.

In some embodiments, the anti-TREM2 antibody is anti-TREM2 monoclonalantibody Ab65. In some embodiments, the anti-TREM2 antibody is anisolated antibody which binds essentially the same TREM2 epitope asAb65. In some embodiments, the anti-TREM2 antibody is an isolatedantibody comprising the HVR-H1, HVR-H2, and HVR-H3 of the heavy chainvariable domains of monoclonal antibody Ab65. In some embodiments, theanti-TREM2 antibody is an isolated antibody comprising the HVR-L1,HVR-L2, and HVR-L3 of the light chain variable domains of monoclonalantibody Ab65. In some embodiments, the anti-TREM2 antibody is anisolated antibody comprising the HVR-H1, HVR-H2, and HVR-H3 of the heavychain variable domains and the HVR-L1, HVR-L2, and HVR-L3 of the lightchain variable domains of monoclonal antibody Ab65. In certainembodiments, the anti-TREM2 antibody is an agonist antibody. In otherembodiments, the anti-TREM2 antibody is an antagonist antibody. In someembodiments, anti-TREM2 antibodies of the present disclosure comprise atleast one, two, three, four, five, or six HVRs selected from (i) HVR-H1comprising the amino acid sequence of SEQ ID NO:9, or an amino acidsequence with at least about 95% homology to the amino acid sequence ofSEQ ID NO:9; (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO: 34, or an amino acid sequence with at least about 95% homology tothe amino acid sequence of SEQ ID NO:34; (iii) HVR-H3 comprising theamino acid sequence of SEQ ID NO: 101, or an amino acid sequence with atleast about 95% homology to the amino acid sequence of SEQ ID NO:101;(iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 124 or anamino acid sequence with at least about 95% homology to the amino acidsequence of SEQ ID NO: 124; (v) HVR-L2 comprising the amino acidsequence of SEQ ID NO: 144 or an amino acid sequence with at least about95% homology to the amino acid sequence of SEQ ID NO: 144; and (vi)HVR-L3 comprising the amino acid sequence of SEQ ID NO:215, or an aminoacid sequence with at least about 95% homology to the amino acidsequence of SEQ ID NO:215.

In some embodiments, anti-TREM2 antibodies of the present disclosurebind to one or more amino acids within amino acid residues 43-50 ofhuman TREM 2 (SEQ ID NO: 1), or within amino acid residues on a TREM2protein corresponding to amino acid residues 43-50 of SEQ ID NO: 1. Insome embodiments, anti-TREM2 antibodies of the present disclosure bindto one or more amino acids within amino acid residues 49-57 of humanTREM 2 (SEQ ID NO: 1), or within amino acid residues on a TREM2 proteincorresponding to amino acid residues 49-57 of SEQ ID NO: 1. In someembodiments, anti-TREM2 antibodies of the present disclosure bind to anepitope that includes one or more amino acid residues within amino acidresidues 43-50 of human TREM 2 (SEQ ID NO: 1). In some embodiments,anti-TREM2 antibodies of the present disclosure bind to an epitope thatincludes one or more amino acid residues within amino acid residues49-57 of human TREM 2 (SEQ ID NO: 1). In some embodiments, anti-TREM2antibodies of the present disclosure competitively inhibit binding of atleast one of the following anti-TREM2 antibodies: Ab21 and Ab52. In someembodiments, anti-TREM2 antibodies of the present disclosure bind to anepitope of human TREM2 that is the same as or overlaps with the TREM2epitope bound by at least one of the following anti-TREM2 antibodies:Ab21 and Ab52. In some embodiments, anti-TREM2 antibodies of the presentdisclosure comprise (a) a heavy chain variable region comprising atleast one, two, or three HVRs selected from HVR-H1, HVR-H2, and HVR-H3of any one of antibodies Ab21 and Ab52; and/or (b) a light chainvariable region comprising at least one, two, or three HVRs selectedfrom HVR-L1, HVR-L2, and HVR-L3 of any one of antibodies Ab21 and Ab52.In some embodiments, the HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, andHVR-L3 comprise Kabat CDR, Chothia CDR, or Contact CDR sequences asshown in Table 1 and/or Table 8. In some embodiments, anti-TREM2antibodies of the present disclosure comprise a heavy chain variableregion of any one of antibodies Ab21 and Ab52; and/or a light chainvariable region of any one of antibodies Ab21 and Ab52. In someembodiments, the anti-TREM2 antibody is anti-TREM2 monoclonal antibodyAb52 or Ab21. In some embodiments, the anti-TREM2 antibody is anisolated antibody which binds essentially the same TREM2 epitope as Ab52or Ab21. In certain embodiments, the anti-TREM2 antibody is an agonistantibody. In other embodiments, the anti-TREM2 antibody is an antagonistantibody.

In some embodiments, the anti-TREM2 antibody is an isolated antibodycomprising the HVR-H1, HVR-H2, and HVR-H3 of the heavy chain variabledomains of monoclonal antibody Ab52 or Ab21. In some embodiments, theanti-TREM2 antibody is an isolated antibody comprising the HVR-L1,HVR-L2, and HVR-L3 of the light chain variable domains of monoclonalantibody Ab52 or Ab21. In some embodiments, the anti-TREM2 antibody isan isolated antibody comprising the HVR-H1, HVR-H2, and HVR-H3 of theheavy chain variable domains and the HVR-L1, HVR-L2, and HVR-L3 of thelight chain variable domains of monoclonal antibody Ab52 or Ab21. Insome embodiments, anti-TREM2 antibodies of the present disclosurecomprise at least one, two, three, four, five, or six HVRs selected from(i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:398, or anamino acid sequence with at least about 95% homology to the amino acidsequence of SEQ ID NO:398; (ii) HVR-H2 comprising the amino acidsequence of SEQ ID NO: 399, or an amino acid sequence with at leastabout 95% homology to the amino acid sequence of SEQ ID NO:399; (iii)HVR-H3 comprising the amino acid sequence of SEQ ID NO:400, or an aminoacid sequence with at least about 95% homology to the amino acidsequence of SEQ ID NO:400; (iv) HVR-L1 comprising the amino acidsequence of SEQ ID NO:401 or an amino acid sequence with at least about95% homology to the amino acid sequence of SEQ ID NO:401; (v) HVR-L2comprising the amino acid sequence of SEQ ID NO:402 or an amino acidsequence with at least about 95% homology to the amino acid sequence ofSEQ ID NO:402; and (vi) HVR-L3 comprising the amino acid sequence of SEQID NO:403, or an amino acid sequence with at least about 95% homology tothe amino acid sequence of SEQ ID NO:403. In some embodiments,anti-TREM2 antibodies of the present disclosure comprise at least one,two, three, four, five, or six HVRs selected from (i) HVR-H1 comprisingthe amino acid sequence of SEQ ID NO:404, or an amino acid sequence withat least about 95% homology to the amino acid sequence of SEQ ID NO:404;(ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 405, or anamino acid sequence with at least about 95% homology to the amino acidsequence of SEQ ID NO:405; (iii) HVR-H3 comprising the amino acidsequence of SEQ ID NO:406, or an amino acid sequence with at least about95% homology to the amino acid sequence of SEQ ID NO:406; (iv) HVR-L1comprising the amino acid sequence of SEQ ID NO:407, or an amino acidsequence with at least about 95% homology to the amino acid sequence ofSEQ ID NO:407; (v) HVR-L2 comprising the amino acid sequence of SEQ IDNO:408, or an amino acid sequence with at least about 95% homology tothe amino acid sequence of SEQ ID NO:408; and (vi) HVR-L3 comprising theamino acid sequence of SEQ ID NO:409, or an amino acid sequence with atleast about 95% homology to the amino acid sequence of SEQ ID NO:409.

In some embodiments, anti-TREM2 antibodies of the present disclosurecomprise a heavy chain variable region, and the heavy chain variableregion comprises at least one, at least two, or three HVR sequencesselected from those listed in Table land/or Table 8; and/or comprise alight chain variable region, and the light chain variable regioncomprises at least one, at least two, or three HVR sequences selectedfrom those listed in Table land/or Table 8.

In some embodiments, anti-TREM2 antibodies of the present disclosurecompete for binding of TREM2 with one or more TREM2 ligands. Examples ofsuitable TREM2 ligands include, without limitation, TREM2 ligandsexpressed by E. coli cells, apoptotic cells, nucleic acids, anioniclipids, zwitterionic lipids, negatively charged phospholipids,phosphatidylserine, sulfatides, phosphatidylcholin, sphingomyelin,membrane phospholipids, lipidated proteins, proteolipids, lipidatedpeptides, and lipidated amyloid beta peptide. Accordingly, in certainembodiments, the one or more TREM2 ligands comprise E. coli cells,apoptotic cells, nucleic acids, anionic lipids, zwitterionic lipids,negatively charged phospholipids, phosphatidylserine, sulfatides,phosphatidylcholin, sphingomyelin, membrane phospholipids, lipidatedproteins, proteolipids, lipidated peptides, and/or lipidated amyloidbeta peptide. In certain embodiments, the anti-TREM2 antibody is anagonist antibody. In other embodiments, the anti-TREM2 antibody is anantagonist antibody.

The dissociation constants (K_(D)) of anti-TREM2 antibodies for humanTREM2 (e.g., human TREM2-Fc fusion proteins and human monomeric TREM2proteins) and mouse TREM2 (e.g., mouse TREM2-Fc fusion proteins) may beless than 10 nM, less than 9.5 nM, less than 9 nM, less than 8.5 nM,less than 8 nM, less than 7.5 nM, less than 7 nM, less than 7 nM, lessthan 6.9 nM, less than 6.8 nM, less than 6.7 nM, less than 6.6 nM, lessthan 6.5 nM, less than 6.4 nM, less than 6.3 nM, less than 6.2 nM, lessthan 6.1 nM, less than 6 nM, less than 5.9 nM, less than 5.8 nM, lessthan 5.75 nM, less than 5.7 nM, less than 5.6 nM, less than 5.5 nM, lessthan 5.4 nM, less than 5.3 nM, less than 5.2 nM, less than 5.1 nM, lessthan 5 nM, less than 4.5 nM, less than 4 nM, less than 3.5 nM, less than3 nM, less than 2.5 nM, less than 2 nM, less than 1.5 nM, less than 1nM, less than 0.95 nM, less than 0.9 nM, less than 0.85 nM, less than0.8 nM, less than 0.75 nM, less than 0.7 nM, less than 0.65 nM, lessthan 0.6 nM, less than 0.55 nM, less than 0.5 nM, less than 0.45 nM,less than 0.4 nM, less than 0.35 nM, less than 0.3 nM, less than 0.29nM, less than 0.28 nM, less than 0.27 nM, less than 0.26 nM, less than0.25 nM, less than 0.24 nM, less than 0.23 nM, less than 0.22 nM, lessthan 0.21 nM, less than 0.2 nM, less than 0.15 nM, less than 0.1 nM,less than 0.095 nM, less than 0.09 nM, less than 0.085 nM, less than0.08 nM, less than 0.075 nM, less than 0.07 nM, less than 0.065 nM, lessthan 0.06 nM, less than 0.055 nM, or less than 0.05 nM. In someembodiments, dissociation constants range from less than about 5.75 nMto less than about 0.09 nM. In some embodiments, dissociation constantsof anti-TREM2 antibodies for human TREM2-Fc fusion proteins range fromless than about 1.51 nM to less than about 0.35 nM. In some embodiments,dissociation constants of anti-TREM2 antibodies for human monomericTREM2 proteins range from less than about 5.75 nM to less than about1.15 nM. In some embodiments, dissociation constants of anti-TREM2antibodies for mouse TREM2-Fc fusion proteins range from less than about0.23 nM to less than about 0.09 nM. In some embodiments, dissociationconstants range from less than about 6.70 nM to less than about 0.23 nM.In some embodiments, dissociation constants of anti-TREM2 antibodies forhuman TREM2-Fc fusion proteins range from less than about 0.71 nM toless than about 0.23 nM. In some embodiments, dissociation constants ofanti-TREM2 antibodies for human monomeric TREM2 proteins range from lessthan about 6.70 nM to less than about 0.66 nM. In some embodiments,dissociation constants of anti-TREM2 antibodies for mouse TREM2-Fcfusion proteins range from less than about 4.90 nM to less than about0.35 nM. Dissociation constants may be determined through any analyticaltechnique, including any biochemical or biophysical technique such asELISA, surface plasmon resonance (SPR), bio-layer interferometry (see,e.g., Octet System by ForteBio), isothermal titration calorimetry (ITC),differential scanning calorimetry (DSC), circular dichroism (CD),stopped-flow analysis, and colorimetric or fluorescent protein meltinganalyses. In certain embodiments, the anti-TREM2 antibody is an agonistantibody. In other embodiments, the anti-TREM2 antibody is an antagonistantibody.

Additional anti-TREM2 antibodies, e.g., antibodies that specificallybind to a TREM2 protein of the present disclosure, may be identified,screened, and/or characterized for their physical/chemical propertiesand/or biological activities by various assays known in the art.

Anti-DAP12 Antibodies

Certain aspects of the present disclosure related to anti-DAP12antibodies.

Anti-DAP12 antibodies of the present disclosure generally bind to one ormore DAP12 proteins expressed in a cell. In certain embodiments theDAP12 protein is expressed on a cell surface.

In some embodiments, anti-DAP12 antibodies of the present disclosure areagonist antibodies or antagonist antibodies that bind to a DAP12 proteinof the present disclosure expressed on the surface of a cell andmodulate (e.g., induce or inhibit) at least one DAP12 activity of thepresent disclosure after binding to the surface-expressed DAP12 protein.In some embodiments, anti-DAP12 antibodies of the present disclosure areinert antibodies.

In certain embodiments the DAP12 protein is expressed on a cell surface.In some embodiment anti-DAP12 antibodies of the present disclosuremodulate (e.g., induce or inhibit) one or more DAP12 activities. TheDAP12 activities modulated (e.g., induced or inhibited) by theanti-DAP12 antibodies may include, without limitation, binding to TREM2;DAP12 phosphorylation; TREM2 phosphorylation; recruitment of Syk, ZAP70,or both to a DAP12/TREM2 complex; PI3K activation; increased expressionof anti-inflammatory mediators (e.g. cytokines); reduced expression ofpro-inflammatory mediators; ERK phosphorylation; increased expression ofCCR7, induction of microglial cell chemotaxis toward CCL19 and CCL21expressing cells; enhancement, normalization, or both of the ability ofbone marrow-derived dendritic cells to induce antigen-specific T-cellproliferation; induction of osteoclast production, increased rate ofosteoclastogenesis, or both; increased survival and function ofmicroglial cells and/or macrophages (such as M1 macrophages and/ormicroglial cells, activated M1 macrophages and/or microglial cells,and/or M2 macrophages and/or microglial cells), dendritic cells,monocytes, osteoclasts, Langerhans cells of skin, and/or Kupffer cells;induction of apoptotic neuron clearance; reduced expression of TNF-α;SYK phosphorylation; increased expression of CD83 and/or CD86 ondendritic cells, monocytes, macrophages, and/or microglia; reducedsecretion of one or more inflammatory cytokines (such as TNF-α, IL-10,IL-6, and/or MCP-1); reduced expression of one or more inflammatoryreceptors (such as CD86); increased phagocytosis by macrophages,dendritic cells, monocytes, and/or microglia under conditions of reducedlevels of MCSF; reduced phagocytosis by macrophages, dendritic cells,monocytes, and/or microglia in the presence of normal levels of MCSF;and/or increased activity of one or more TREM2-dependent genes (e.g.,transcription factors of the nuclear factor of activated T-cells (NFAT)family of transcription factors). The anti-TREM2 antibodies of thepresent disclosure can be used to prevent, reduce risk of, or treatdementia, frontotemporal dementia, Alzheimer's disease, Nasu-Hakoladisease, and multiple sclerosis. In some embodiments, the anti-TREM2antibodies of the present disclosure are monoclonal antibodies.Anti-TREM2 antibodies of the present disclosure may be tested forinducing one or more TREM2 activities (e.g., DAP12 phosphorylation;recruitment of Syk, ZAP70, or both to DAP12; PI3K activation; increasedexpression of anti-inflammatory mediators; reduced expression ofpro-inflammatory mediators; ERK phosphorylation; increased expression ofCCR7, induction of microglial cell chemotaxis toward CCL19 and CCL21expressing cells; enhancement, normalization, or both of the ability ofbone marrow-derived dendritic cells to induce antigen-specific T-cellproliferation; induction of osteoclast production, increased rate ofosteoclastogenesis, or both; induction of apoptotic neuron clearance;reduced expression of TNF-α; SYK phosphorylation; increased expressionof CD83 and/or CD86 on dendritic cells; reduced secretion of one or moreinflammatory cytokines; reduced expression of one or more inflammatoryreceptors; increased survival and/or function of dendritic cells,macrophages and/or microglial cells; increased phagocytosis bymacrophages, dendritic cells, and/or microglia under conditions ofreduced levels of MCSF; reduced phagocytosis by macrophages, dendriticcells, and/or microglia in the presence of normal levels of MCSF, andincreased activity of one or more TREM2-dependent genes (e.g.,transcription factors of the nuclear factor of activated T-cells (NFAT)family of transcription factors) using any suitable method known in theart and/or described herein. For example, the anti-TREM2 antibodies canbe assayed in vitro for tyrosine phosphorylation of TREM2, DAP12 and/orERK, by assaying for recruitment of Syk and/or ZAP70 to a Dap12/TREM2complex, by assaying for PI3K activation, by assaying for induction ofexpression of anti-inflammatory mediators (e.g., IL-12p70, IL-6, andIL-10) or CCR7, or by assaying for reduced expression ofpro-inflammatory mediators (e.g., IL1-β and TNF) with TLR stimulation(e.g., LPS, CpG DNA, or Zymosan). Useful assays may include westernblots (e.g., for tyrosine-phosphorylated DAP12 orthreonine/serine-phosphorylated PI3K-kinase substrates), ELISA (e.g.,for secreted interleukin or cytokine secretion), FACS (e.g., foranti-TREM2 binding to TREM2), immunocytochemistry (e.g., for e.g., fortyrosine-phosphorylated DAP12 or threonine/serine-phosphorylatedPI3K-kinase substrates), reporter-gene assays (e.g., for TLRactivation), increased survival and/or function of microglial cells,dendritic cells, monocytes, and/or macrophages, increased phagocytosisof apoptotic neurons, damaged synapses, A beta, and/or other cellulardebris by macrophages, dendritic cells, osteoclasts, and/or microglialcells, increased cytoskeleton reorganization, and decreased microglialpro-inflammatory responses, or other assays known in the art.

Certain aspects of the present disclosure provide anti-DAP12 antibodiesthat bind to a human DAP12, or a homolog thereof, including withoutlimitation a mammalian DAP12 protein, mouse DAP12 protein (UniprotAccession No. Q99NH8), rat DAP12 protein (Uniprot Accession No. D3ZZ89),Rhesus monkey DAP12 protein (Uniprot Accession No. F6QVF2), bovine DAP12protein (Uniprot Accession No. Q05B59), equine DAP12 protein (UniprotAccession No. F7D6L0), pig DAP12 protein (Uniprot Accession No. H2EZZ3),and dog DAP12 protein (Uniprot Accession No. E2RP46); and induce atleast one DAP12 activity. In some embodiments, the at least one DAP12activity is DAP12 phosphorylation, TREM2 phosphorylation, PI3Kactivation, increased expression of one or more anti-inflammatorymediators, and/or reduced expression of one or more pro-inflammatorymediators.

In some embodiments, anti-DAP12 antibodies of the present disclosurebind to a DAP12 protein of the present disclosure and/or naturallyoccurring variants. In certain preferred embodiments, the anti-DAP12antibodies bind to human DAP12.

In some embodiments, anti-DAP12 antibodies of the present disclosurebind to a DAP12 protein of the present disclosure expressed on thesurface of a cell and induce at least one DAP12 activity of the presentdisclosure after binding to the surface expressed DAP12 protein.

In certain embodiments, anti-DAP12 antibodies of the present disclosurebind to one or more amino acids within amino acid residues 22-40 ofhuman DAP12 (SEQ ID NO: 2), or within amino acid residues on a DAP12protein corresponding to amino acid residues 22-40 of SEQ ID NO: 2.

The dissociation constants (K_(D)) of anti-DAP12 antibodies for humanDAP12 and mouse DAP12 may be less than 10 nM, less than 9.5 nM, lessthan 9 nM, less than 8.5 nM, less than 8 nM, less than 7.5 nM, less than7 nM, less than 6.5 nM, less than 6 nM, less than 5.9 nM, less than 5.8nM, less than 5.75 nM, less than 5.7 nM, less than 5.6 nM, less than 5.5nM, less than 5.4 nM, less than 5.3 nM, less than 5.2 nM, less than 5.1nM, less than 5 nM, less than 4.5 nM, less than 4 nM, less than 3.5 nM,less than 3 nM, less than 2.5 nM, less than 2 nM, less than 1.5 nM, lessthan 1 nM, less than 0.95 nM, less than 0.9 nM, less than 0.85 nM, lessthan 0.8 nM, less than 0.75 nM, less than 0.7 nM, less than 0.65 nM,less than 0.6 nM, less than 0.55 nM, less than 0.5 nM, less than 0.45nM, less than 0.4 nM, less than 0.35 nM, less than 0.3 nM, less than0.25 nM, less than 0.2 nM, less than 0.15 nM, less than 0.1 nM, lessthan 0.095 nM, less than 0.09 nM, less than 0.085 nM, less than 0.08 nM,less than 0.075 nM, less than 0.07 nM, less than 0.065 nM, less than0.06 nM, less than 0.055 nM, or less than 0.05 nM. Dissociationconstants may be determined through any analytical technique, includingany biochemical or biophysical technique such as ELISA, surface plasmonresonance (SPR), bio-layer interferometry (see, e.g., Octet System byForteBio), isothermal titration calorimetry (ITC), differential scanningcalorimetry (DSC), circular dichroism (CD), stopped-flow analysis, andcolorimetric or fluorescent protein melting analyses.

Additional anti-DAP12 antibodies, e.g., antibodies that specificallybind to a DAP12 protein of the present disclosure, may be identified,screened, and/or characterized for their physical/chemical propertiesand/or biological activities by various assays known in the art.

Bispecific Antibodies

Certain aspects of the present disclosure relate to bispecificantibodies that bind to both TREM2 and DAP12 proteins of the presentdisclosure. Methods of generating bispecific antibodies are well knownin the art and described herein. In some embodiments, bispecificantibodies of the present disclosure bind to one or more amino acidresidues of human TREM2 (SEQ ID NO: 1), or amino acid residues on aTREM2 protein corresponding to amino acid residues of SEQ ID NO: 1. Inother embodiments, bispecific antibodies of the present disclosure alsobind to one or more amino acid residues of human DAP12 (SEQ ID NO: 2),or amino acid residues on a DAP12 protein corresponding to amino acidresidues of SEQ ID NO: 2.

In some embodiments, bispecific antibodies of the present disclosurerecognize a first antigen and a second antigen. In some embodiments, thefirst antigen is human TREM2 or a naturally occurring variant thereof,or human DAP12 or a naturally occurring variant thereof. In someembodiments, the second antigen is a disease-causing protein selectedfrom amyloid beta or fragments thereof, Tau, IAPP, alpha-synuclein,TDP-43, FUS protein, prion protein, PrPSc, huntingtin, calcitonin,superoxide dismutase, ataxin, Lewy body, atrial natriuretic factor,islet amyloid polypeptide, insulin, apolipoprotein AI, serum amyloid A,medin, prolactin, transthyretin, lysozyme, beta 2 microglobulin,gelsolin, keratoepithelin, cystatin, immunoglobulin light chain AL,S-IBM protein, Repeat-associated non-ATG (RAN) translation products,DiPeptide repeat (DPR) peptides, glycine-alanine (GA) repeat peptides,glycine-proline (GP) repeat peptides, glycine-arginine (GR) repeatpeptides, proline-alanine (PA) repeat peptides, and proline-arginine(PR) repeat peptides. In some embodiments, the second antigen is a bloodbrain barrier targeting protein selected from trasnferin receptor,insulin receptor, insulin like growth factor receptor, LRP-1, and LRP1;or ligands and/or proteins expressed on immune cells, wherein theligands and/or proteins selected from the group consisting of: CD40,OX40, ICOS, CD28, CD137/4-1BB, CD27, GITR, PD-L1, CTLA4, PD-L2, PD-1,B7-H3, B7-H4, HVEM, BTLA, KIR, GAL9, TIM3, A2AR, LAG, andphosphatidylserine. Alternatively, the second antigen may be, withoutlimitation, a protein expressed on one or more tumor cells.

Antibody Fragments

Certain aspects of the present disclosure relate to antibody fragmentsthat bind to one or more human proteins selected from human TREM2, anaturally occurring variant of human TREM2, a disease variant of humanTREM2, human DAP12, and naturally occurring variant of human DAP12. Insome embodiments, the antibody fragment is an Fab, Fab′, Fab′-SH,F(ab′)2, Fv or scFv fragment. In some embodiments, the antibody fragmentis used in combination with one or more antibodies that specificallybind a disease-causing protein selected from: amyloid beta or fragmentsthereof, Tau, IAPP, alpha-synuclein, TDP-43, FUS protein, prion protein,PrPSc, huntingtin, calcitonin, superoxide dismutase, ataxin, Lewy body,atrial natriuretic factor, islet amyloid polypeptide, insulin,apolipoprotein AI, serum amyloid A, medin, prolactin, transthyretin,lysozyme, beta 2 microglobulin, gelsolin, keratoepithelin, cystatin,immunoglobulin light chain AL, S-IBM protein, Repeat-associated non-ATG(RAN) translation products, DiPeptide repeat (DPR) peptides,glycine-alanine (GA) repeat peptides, glycine-proline (GP) repeatpeptides, glycine-arginine (GR) repeat peptides, proline-alanine (PA)repeat peptides, proline-arginine (PR) repeat peptides, and anycombination thereof, and/or one or more antibodies that specificallybind a cancer-associated protein selected from: CD40, OX40, ICOS, CD28,CD137/4-1BB, CD27, GITR, PD-L1, CTLA4, PD-L2, PD-1, B7-H3, B7-H4, HVEM,BTLA, KIR, GAL9, TIM3, A2AR, LAG, phosphatidylserine, and anycombination thereof.

Antibody Frameworks

Any of the antibodies described herein further include a framework. Insome embodiments, the framework is a human immunoglobulin framework. Forexample, in some embodiments, an antibody (e.g., an anti-TREM2 antibody)comprises HVRs as in any of the above embodiments and further comprisesan acceptor human framework, e.g., a human immunoglobulin framework or ahuman consensus framework. Human immunoglobulin frameworks may be partof the human antibody, or a non-human antibody may be humanized byreplacing one or more endogenous frameworks with human frameworkregion(s). Human framework regions that may be used for humanizationinclude but are not limited to: framework regions selected using the“best-fit” method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993));framework regions derived from the consensus sequence of humanantibodies of a particular subgroup of light or heavy chain variableregions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285(1992); and Presta et al. J. Immunol., 151:2623 (1993)); human mature(somatically mutated) framework regions or human germline frameworkregions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633(2008)); and framework regions derived from screening FR libraries (see,e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok etal., J. Biol. Chem. 271:22611-22618 (1996)).

In some embodiments, an antibody comprises a heavy chain variable regioncomprising an HVR-H1, an HVR-H2, and an HVR-H3 of the present disclosureand one, two, three or four of the heavy chain framework regions asshown in FIG. 2B and/or FIG. 20A. In some embodiments, an antibodycomprises a light chain variable region comprising an HVR-L1, an HVR-L2,and an HVR-L3 of the present disclosure and one, two, three or four ofthe light chain framework regions as shown in FIG. 2C and/or FIG. 20B.In some embodiments, an antibody comprises a heavy chain variable regioncomprising an HVR-H1, an HVR-H2, and an HVR-H3 of the present disclosureand one, two, three or four of the heavy chain framework regions asshown in FIG. 2B and/or FIG. 20A and further comprises a light chainvariable region comprising an HVR-L1, an HVR-L2, and an HVR-L3 of thepresent disclosure and one, two, three or four of the light chainframework regions as shown in FIG. 2C and/or FIG. 20B.

TREM2 and/or DAP12 Binding and Phosphorylation

In some embodiments, the anti-TREM2 and/or anti-DAP12 antibodies of thepresent disclosure may induce binding of TREM2 to DAP12 and/or DAP12binding to TREM2. In other embodiments, the anti-TREM2 and/or anti-DAP12antibodies of the present disclosure may induce TREM2 phosphorylationafter binding to a TREM2 and/or DAP12 protein expressed in a cell. Inother embodiments, the anti-TREM2 and/or anti-DAP12 antibodies of thepresent disclosure may induce DAP12 phosphorylation after binding to aTREM2 and/or DAP12 protein expressed in a cell. In other embodiments,TREM2-mediated TREM2 and/or DAP12 phosphorylation is induced by one ormore SRC family tyrosine kinases. Examples of Src family tyrosinekinases include, without limitation, Src, Yes, Fyn, Fgr, Lck, Hck, Blk,Lyn, and Frk.

DAP12 is variously referred to as TYRO protein tyrosine kinase-bindingprotein, TYROBP, KARAP, and PLOSL. DAP12 is a transmembrane signalingprotein that contains an immunoreceptor tyrosine-based activation motif(ITAM) in its cytoplasmic domain. In certain embodiments, the anti-TREM2and/or anti-DAP12 antibody may induce DAP12 phosphorylation in its ITAMmotif. Any method known in the art for determining proteinphosphorylation, such as DAP12 phosphorylation, may be used.

In some embodiments, DAP12 is phosphorylated by SRC family kinases,resulting in the recruitment and activation of the Syk kinase, ZAP70kinase, or both, to DAP12. Thus, in certain embodiments, the anti-TREM2and/or anti-DAP12 antibodies of the present disclosure may recruit Syk,ZAP70, or both to a DAP12/TREM2 complex.

Without wishing to be bound by theory, it is believed that anti-TREM2and/or anti-DAP12 antibodies of the present disclosure are useful forpreventing, lowering the risk of, or treating conditions and/or diseasesassociated with decreased levels of DAP12 activity, DAP12phosphorylation, or recruitment of Syk, ZAP70, or both to a DAP12/TREM2complex, including dementia, frontotemporal dementia, Alzheimer'sdisease, Nasu-Hakola disease, Parkinson's disease, Amyotrophic lateralsclerosis, Huntington's disease, Taupathy disease, and/or multiplesclerosis.

PI3K Activation

In some embodiments, the anti-TREM2 and/or anti-DAP12 antibodies of thepresent disclosure may induce PI3K activation after binding to a TREM2and/or DAP12 protein expressed in a cell.

PI3Ks are a family of related intracellular signal transducer kinasescapable of phosphorylating the 3-position hydroxyl group of the inositolring of phosphatidylinositol (PtdIns). The PI3K family is divided intothree different classes (Class I, Class II, and Class III) based onprimary structure, regulation, and in vitro lipid substrate specificity.

Activated PI3K produces various 3-phosphorylated phosphoinositides,including without limitation, PtdIns3P, PtdIns(3,4)P2, PtdIns(3,5)P2,and PtdIns(3,4,5)P3. These 3-phosphorylated phosphoinositides functionin a mechanism by which signaling proteins are recruited to variouscellular membranes. These signaling proteins containphosphoinositide-binding domains, including without limitation, PXdomains, pleckstrin homology domains (PH domains), and FYVE domains. Anymethod known in the art for determining PI3K activation may be used.

Without wishing to be bound by theory, it is believed that anti-TREM2and/or anti-DAP12 antibodies of the present disclosure are beneficialfor preventing, lowering the risk of, or treating conditions and/ordiseases associated with decreased levels of PI3K activity, includingdementia, frontotemporal dementia, Alzheimer's disease, Nasu-Hakoladisease, Parkinson's disease, Amyotrophic lateral sclerosis,Huntington's disease, Taupathy disease, and/or multiple sclerosis.

Increased Expression of Anti-Inflammatory Mediators

In some embodiments, the anti-TREM2 and/or anti-DAP12 antibodies of thepresent disclosure have an anti-inflammatory activity in the brain afterbinding to a TREM2 and/or DAP12 protein expressed on a cell surface. Ithas recently been reported that TREM2 has an anti-inflammatory role inthe brain. In certain embodiments, the anti-TREM2 and/or anti-DAP12antibodies of the present disclosure increase the expression ofanti-inflammatory mediators (e.g., cytokines) and/or reduce theexpression of pro-inflammatory mediators after binding to a TREM2 and/orDAP12 protein expressed in a cell.

Inflammation is part of a complex biological response of vasculartissues to harmful stimuli, such as pathogens, damaged cells, andirritants. The classical signs of acute inflammation are pain, heat,redness, swelling, and loss of function. Inflammation is a protectiveattempt by an organism to remove the injurious stimuli and to initiatethe healing process. Inflammation can be classified as either acuteinflammation or chronic inflammation. Acute inflammation is the initialresponse of the body to harmful stimuli and is achieved by the increasedmovement of plasma and leukocytes (especially granulocytes) from theblood into the injured tissues. A cascade of biochemical eventspropagates and matures the inflammatory response, involving the localvascular system, the immune system, and various cells within the injuredtissue. Chronic inflammation is prolonged inflammation that leads to aprogressive shift in the type of cells present at the site ofinflammation and is characterized by simultaneous destruction andhealing of the tissue from the inflammatory process.

As used herein, anti-inflammatory mediators are proteins involved eitherdirectly or indirectly (e.g., by way of an anti-inflammatory signalingpathway) in a mechanism that reduces, inhibits, or inactivates aninflammatory response. Any method known in the art for identifying andcharacterizing anti-inflammatory mediators may be used. Examples ofanti-inflammatory mediators include, without limitation, cytokines, suchas IL-12p70, IL-6, and IL-10.

In some embodiments, the anti-TREM2 and/or anti-DAP12 antibodies of thepresent disclosure may increase expression of anti-inflammatorymediators, such as IL-12p70, IL-6, and IL-10. In certain embodiments,increased expression of the anti-inflammatory mediators occurs inmacrophages, dendritic cells, and/or microglial cells. Increasedexpression may include, without limitation, in increase in geneexpression, an increase in transcriptional expression, or an increase inprotein expression. Any method known in the art for determining gene,transcript (e.g., mRNA), and/or protein expression may be used. Forexample, Northern blot analysis may be used to determineanti-inflammatory mediator gene expression levels, RT-PCR may be used todetermine the level of anti-inflammatory mediator transcription, andWestern blot analysis may be used to determine anti-inflammatorymediator protein levels.

As used herein, an anti-inflammatory mediator may have increasedexpression if its expression in one or more cells of a subject treatedwith an anti-TREM2 and/or anti-DAP12 antibody of the present disclosureis greater than the expression of the same anti-inflammatory mediatorexpressed in one or more cells of a corresponding subject that is nottreated with the anti-TREM2 and/or anti-DAP12 antibody. In someembodiments, an anti-TREM2 and/or anti-DAP12 antibody of the presentdisclosure may increase anti-inflammatory mediator expression in one ormore cells of a subject by at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 100%, at least 110%, at least 115%, at least 120%, at least 125%,at least 130%, at least 135%, at least 140%, at least 145%, at least150%, at least 160%, at least 170%, at least 180%, at least 190%, or atleast 200% for example, as compared to anti-inflammatory mediatorexpression in one or more cells of a corresponding subject that is nottreated with the anti-TREM2 and/or anti-DAP12 antibody. In otherembodiments, an anti-TREM2 and/or anti-DAP12 antibody of the presentdisclosure increases anti-inflammatory mediator expression in one ormore cells of a subject by at least 1.5 fold, at least 1.6 fold, atleast 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2.0 fold,at least 2.1 fold, at least 2.15 fold, at least 2.2 fold, at least 2.25fold, at least 2.3 fold, at least 2.35 fold, at least 2.4 fold, at least2.45 fold, at least 2.5 fold, at least 2.55 fold, at least 3.0 fold, atleast 3.5 fold, at least 4.0 fold, at least 4.5 fold, at least 5.0 fold,at least 5.5 fold, at least 6.0 fold, at least 6.5 fold, at least 7.0fold, at least 7.5 fold, at least 8.0 fold, at least 8.5 fold, at least9.0 fold, at least 9.5 fold, or at least 10 fold, for example, ascompared to anti-inflammatory mediator expression in one or more cellsof a corresponding subject that is not treated with the anti-TREM2and/or anti-DAP12 antibody.

Without wishing to be bound by theory, it is believed that, in someembodiments, anti-TREM2 and/or anti-DAP12 antibodies of the presentdisclosure are useful for preventing, lowering the risk of, or treatingconditions and/or diseases associated with decreased levels of one ormore anti-inflammatory mediators, including dementia, frontotemporaldementia, Alzheimer's disease, Nasu-Hakola disease, Parkinson's disease,Amyotrophic lateral sclerosis, Huntington's disease, Taupathy disease,and/or multiple sclerosis.

Reduced Expression of Pro-Inflammatory Mediators

In some embodiments, the anti-TREM2 and/or anti-DAP12 antibodies of thepresent disclosure may decrease the expression of pro-inflammatorymediators after binding to a TREM2 and/or DAP12 protein expressed in acell.

As used herein, pro-inflammatory mediators are proteins involved eitherdirectly or indirectly (e.g., by way of pro-inflammatory signalingpathways) in a mechanism that induces, activates, promotes, or otherwiseincreases an inflammatory response. Any method known in the art foridentifying and characterizing pro-inflammatory mediators may be used.Examples of pro-inflammatory mediators include, without limitation,cytokines, such as IIFN-a4, IFN-b, IL-1β, TNF-α, IL-10, IL-6, IL-12 p70,IL-8, CRP, TNF, TGF-beta members of the chemokine protein families,IL-20 family members, IL-33, LIF, IFN-gamma, OSM, CNTF, TGF-beta,GM-CSF, IL-11, IL-12, IL-17, IL-18, and CRP.

In some embodiments, the anti-TREM2 and/or anti-DAP12 antibodies of thepresent disclosure may decrease functional expression and/or secretionof pro-inflammatory mediators, such as IFN-a4, IFN-b, IL-6, IL-12 p70,IL-1β and TNF, TNF-α, IL-10, IL-8, CRP, TGF-beta members of thechemokine protein families, IL-20 family members, IL-33, LIF, IFN-gamma,OSM, CNTF, TGF-beta, GM-CSF, IL-11, IL-12, IL-17, IL-18, and CRP. Incertain embodiments, decreased expression of the pro-inflammatorymediators occurs in macrophages, dendritic cells, monocytes,osteoclasts, Langerhans cells of skin, Kupffer cells, and/or microglialcells. Decreased expression may include, without limitation, a decreasein gene expression, a decrease in transcriptional expression, or adecrease in protein expression. Any method known in the art fordetermining gene, transcript (e.g., mRNA), and/or protein expression maybe used. For example, Northern blot analysis may be used to determinepro-inflammatory mediator gene expression levels, RT-PCR may be used todetermine the level of pro-inflammatory mediator transcription, andWestern blot analysis may be used to determine pro-inflammatory mediatorprotein levels.

In certain embodiments, pro-inflammatory mediators include inflammatorycytokines. Accordingly, in certain embodiments, the anti-TREM2 and/oranti-DAP12 antibodies of the present disclosure may reduce secretion ofone or more inflammatory cytokines. Examples of inflammatory cytokineswhose secretion may be reduced by the anti-TREM2 and/or anti-DAP12antibodies of the present disclosure include, without limitation, TNF-α,IL-10, IL-6, MCP-1, IFN-a4, IFN-b, IL-1β. IL-8, CRP, TGF-beta members ofthe chemokine protein families, IL-20 family members, IL-33, LIF,IFN-gamma, OSM, CNTF, TGF-beta, GM-CSF, IL-11, IL-12, IL-17, and IL-18.

In certain embodiments, pro-inflammatory mediators include inflammatoryreceptors. Accordingly, in certain embodiments, the anti-TREM2 and/oranti-DAP12 antibodies of the present disclosure may reduce expression ofone or more inflammatory receptors. Examples of inflammatory receptorswhose expression may be reduced by the anti-TREM2 and/or anti-DAP12antibodies of the present disclosure include, without limitation, CD86.

As used herein, a pro-inflammatory mediator may have decreasedexpression if its expression in one or more cells of a subject treatedwith an agonist anti-TREM2 and/or anti-DAP12 antibody of the presentdisclosure is less than the expression of the same pro-inflammatorymediator expressed in one or more cells of a corresponding subject thatis not treated with the agonist anti-TREM2 and/or anti-DAP12 antibody.In some embodiments, the agonist anti-TREM2 and/or anti-DAP12 antibodyof the present disclosure may decrease pro-inflammatory mediatorexpression in one or more cells of a subject by at least 10%, at least15%, at least 20%, at least 25%, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 100%, at least 110%, at least 115%, at least120%, at least 125%, at least 130%, at least 135%, at least 140%, atleast 145%, at least 150%, at least 160%, at least 170%, at least 180%,at least 190%, or at least 200% for example, as compared topro-inflammatory mediator expression in one or more cells of acorresponding subject that is not treated with the agonist anti-TREM2and/or anti-DAP12 antibody. In other embodiments, the agonist anti-TREM2and/or anti-DAP12 antibody may decrease pro-inflammatory mediatorexpression in one or more cells of a subject by at least at least 1.5fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least1.9 fold, at least 2.0 fold, at least 2.1 fold, at least 2.15 fold, atleast 2.2 fold, at least 2.25 fold, at least 2.3 fold, at least 2.35fold, at least 2.4 fold, at least 2.45 fold, at least 2.5 fold, at least2.55 fold, at least 3.0 fold, at least 3.5 fold, at least 4.0 fold, atleast 4.5 fold, at least 5.0 fold, at least 5.5 fold, at least 6.0 fold,at least 6.5 fold, at least 7.0 fold, at least 7.5 fold, at least 8.0fold, at least 8.5 fold, at least 9.0 fold, at least 9.5 fold, or atleast 10 fold, for example, as compared to pro-inflammatory mediatorexpression in one or more cells of a corresponding subject that is nottreated with the anti-TREM2 and/or anti-DAP12 antibody.

Without wishing to be bound by theory, it is believed that someanti-TREM2 and/or anti-DAP12 antibodies of the present disclosure may beuseful for preventing, lowering the risk of, or treating conditionsand/or diseases associated with increased levels of one or morepro-inflammatory mediators, including dementia, frontotemporal dementia,Alzheimer's disease, Nasu-Hakola disease, Parkinson's disease,Amyotrophic lateral sclerosis, Huntington's disease, Taupathy disease,and/or multiple sclerosis.

ERK Phosphorylation

In some embodiments, the anti-TREM2 and/or anti-DAP12 antibodies of thepresent disclosure may induce extracellular signal-regulated kinase(ERK) phosphorylation after binding to a TREM2 and/or DAP12 proteinexpressed in a cell.

Extracellular-signal-regulated kinases (ERKs) are widely expressedprotein kinase intracellular signaling kinases that are involved in, forexample, the regulation of meiosis, mitosis, and postmitotic functionsin differentiated cells. Various stimuli, such as growth factors,cytokines, virus infection, ligands for heterotrimeric G protein-coupledreceptors, transforming agents, and carcinogens, activate ERK pathways.Phosphorylation of ERKs leads to the activation of their kinaseactivity.

Without wishing to be bound by theory, it is believed that anti-TREM2and/or anti-DAP12 antibodies of the present disclosure are beneficialfor preventing, lowering the risk of, or treating conditions and/ordiseases associated with decreased levels of ERK phosphorylation,including dementia, frontotemporal dementia, Alzheimer's disease,Nasu-Hakola disease, Parkinson's disease, Amyotrophic lateral sclerosis,Huntington's disease, Taupathy disease, and/or multiple sclerosis.

Increased Expression of C-C Chemokine Receptor 7

In some embodiments, the anti-TREM2 and/or anti-DAP12 antibodies of thepresent disclosure may increase expression of C-C chemokine receptor 7(CCR7) after binding to a TREM2 and/or DAP12 protein expressed in acell. Increased expression may include, without limitation, in increasein gene expression, an increase in transcriptional expression, or anincrease in protein expression. Any method known in the art fordetermining gene, transcript (e.g., mRNA), and/or protein expression maybe used. For example, Northern blot analysis may be used to determineanti-inflammatory mediator gene expression levels, RT-PCR may be used todetermine the level of anti-inflammatory mediator transcription, andWestern blot analysis may be used to determine anti-inflammatorymediator protein levels.

C-C chemokine receptor 7 (CCR7) is a member of the G protein-coupledreceptor family. CCR7 is expressed in various lymphoid tissues and canactivate B-cells and T-cells. In some embodiments, CCR7 may modulate themigration of memory T-cells to secondary lymphoid organs, such as lymphnodes. In other embodiments, CCR7 may stimulate dendritic cellmaturation. CCR7 is a receptor protein that can bind the chemokine (C-Cmotif) ligands CCL19/ELC and CCL21.

As used herein, CCR7 may have increased expression if its expression inone or more cells of a subject treated with an anti-TREM2 and/oranti-DAP12 antibody of the present disclosure is greater than theexpression of CCR7 expressed in one or more cells of a correspondingsubject that is not treated with the anti-TREM2 and/or anti-DAP12antibody. In some embodiments, an anti-TREM2 and/or anti-DAP12 antibodyof the present disclosure may increase CCR7 expression in one or morecells of a subject by at least 10%, at least 15%, at least 20%, at least25%, at least 30%, at least 35%, at least 40%, at least 45%, at least50%, at least 55%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least100%, at least 110%, at least 115%, at least 120%, at least 125%, atleast 130%, at least 135%, at least 140%, at least 145%, at least 150%,at least 160%, at least 170%, at least 180%, at least 190%, or at least200% for example, as compared to CCR7 expression in one or more cells ofa corresponding subject that is not treated with the anti-TREM2 and/oranti-DAP12 antibody. In other embodiments, an anti-TREM2 and/oranti-DAP12 antibody of the present disclosure increases CCR7 expressionin one or more cells of a subject by at least 1.5 fold, at least 1.6fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least2.0 fold, at least 2.1 fold, at least 2.15 fold, at least 2.2 fold, atleast 2.25 fold, at least 2.3 fold, at least 2.35 fold, at least 2.4fold, at least 2.45 fold, at least 2.5 fold, at least 2.55 fold, atleast 3.0 fold, at least 3.5 fold, at least 4.0 fold, at least 4.5 fold,at least 5.0 fold, at least 5.5 fold, at least 6.0 fold, at least 6.5fold, at least 7.0 fold, at least 7.5 fold, at least 8.0 fold, at least8.5 fold, at least 9.0 fold, at least 9.5 fold, or at least 10 fold, forexample, as compared to CCR7 expression in one or more cells of acorresponding subject that is not treated with the anti-TREM2 and/oranti-DAP12 antibody.

In some embodiments, increased expression of CCR7 occurs in macrophages,dendritic cells, and/or microglial cells. Increased expression of CCR7may induce microglial cell chemotaxis toward cells expressing thechemokines CCL19 and CCL21. Accordingly, in certain embodiments,anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure mayinduce microglial cell chemotaxis toward CCL19 and CCL21 expressingcells.

Without wishing to be bound by theory, it is believed that, in someembodiments, anti-TREM2 and/or anti-DAP12 antibodies of the presentdisclosure are useful for preventing, lowering the risk of, or treatingconditions and/or diseases associated with decreased levels of CCR7,including dementia, frontotemporal dementia, Alzheimer's disease,Nasu-Hakola disease, Parkinson's disease, Amyotrophic lateral sclerosis,Huntington's disease, Taupathy disease, and/or multiple sclerosis.

Enhancement or Normalization of the Ability of Bone Marrow-DerivedDendritic Cells to Induce Antigen-Specific T-Cell Proliferation

In some embodiments, the anti-TREM2 and/or anti-DAP12 antibodies of thepresent disclosure may enhance and/or normalize the ability of bonemarrow-derived dendritic cells to induce antigen-specific T-cellproliferation after binding to a TREM2 and/or DAP12 protein expressed ina cell.

In some embodiments, agonist anti-TREM2 and/or anti-DAP12 antibodies ofthe present disclosure may enhance and/or normalize the ability of bonemarrow-derived dendritic cells to induce antigen-specific T-cellproliferation in one or more bone marrow-derived dendritic cells of asubject by at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 100%, atleast 110%, at least 115%, at least 120%, at least 125%, at least 130%,at least 135%, at least 140%, at least 145%, at least 150%, at least160%, at least 170%, at least 180%, at least 190%, or at least 200% forexample, as compared to the ability of bone marrow-derived dendriticcells to induce antigen-specific T-cell proliferation in one or morebone marrow-derived dendritic cells of a corresponding subject that isnot treated with the agonist anti-TREM2 and/or anti-DAP12 antibody. Inother embodiments, the agonist anti-TREM2 and/or anti-DAP12 antibody mayenhance and/or normalize the ability of bone marrow-derived dendriticcells to induce antigen-specific T-cell proliferation in one or morebone marrow-derived dendritic cells of a subject by at least at least1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, atleast 1.9 fold, at least 2.0 fold, at least 2.1 fold, at least 2.15fold, at least 2.2 fold, at least 2.25 fold, at least 2.3 fold, at least2.35 fold, at least 2.4 fold, at least 2.45 fold, at least 2.5 fold, atleast 2.55 fold, at least 3.0 fold, at least 3.5 fold, at least 4.0fold, at least 4.5 fold, at least 5.0 fold, at least 5.5 fold, at least6.0 fold, at least 6.5 fold, at least 7.0 fold, at least 7.5 fold, atleast 8.0 fold, at least 8.5 fold, at least 9.0 fold, at least 9.5 fold,or at least 10 fold, for example, as compared to the ability of bonemarrow-derived dendritic cells to induce antigen-specific T-cellproliferation in one or more bone marrow-derived dendritic cells of acorresponding subject that is not treated with the agonist anti-TREM2and/or anti-DAP12 antibody.

Without wishing to be bound by theory, it is believed that anti-TREM2and/or anti-DAP12 antibodies of the present disclosure are beneficialfor preventing, lowering the risk of, or treating conditions and/ordiseases associated with an increased or disregulated ability of bonemarrow-derived dendritic cells to induce antigen-specific T-cellproliferation, including dementia, frontotemporal dementia, Alzheimer'sdisease, Nasu-Hakola disease, Parkinson's disease, Amyotrophic lateralsclerosis, Huntington's disease, Taupathy disease, and/or multiplesclerosis.

Osteoclast Production

In some embodiments, the anti-TREM2 and/or anti-DAP12 antibodies of thepresent disclosure may induce osteoclast production and/or increase therate of osteoclastogenesis after binding to a TREM2 and/or DAP12 proteinexpressed in a cell.

As used herein, an osteoclast is a type of bone cell that can removebone tissue by removing its mineralized matrix and breaking up theorganic bone (e.g., bone resorption). Osteoclasts can be formed by thefusion of cells of the monocyte-macrophage cell line. In someembodiments, osteoclasts may be characterized by high expression oftartrate resistant acid phosphatase (TRAP) and cathepsin K.

As used herein, the rate of osteoclastogenesis may be increased if therate of osteoclastogenesis in a subject treated with an agonistanti-TREM2 and/or anti-DAP12 antibody of the present disclosure isgreater than the rate of osteoclastogenesis in a corresponding subjectthat is not treated with the agonist anti-TREM2 and/or anti-DAP12antibody. In some embodiments, an agonist anti-TREM2 and/or anti-DAP12antibody of the present disclosure may increase the rate ofosteoclastogenesis in a subject by at least 10%, at least 15%, at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least45%, at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 100%, at least 110%, at least 115%, at least 120%, atleast 125%, at least 130%, at least 135%, at least 140%, at least 145%,at least 150%, at least 160%, at least 170%, at least 180%, at least190%, or at least 200% for example, as compared to rate ofosteoclastogenesis in a corresponding subject that is not treated withthe agonist anti-TREM2 and/or anti-DAP12 antibody. In other embodiments,an agonist anti-TREM2 and/or anti-DAP12 antibody of the presentdisclosure may increase the rate of osteoclastogenesis in a subject byat least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8fold, at least 1.9 fold, at least 2.0 fold, at least 2.1 fold, at least2.15 fold, at least 2.2 fold, at least 2.25 fold, at least 2.3 fold, atleast 2.35 fold, at least 2.4 fold, at least 2.45 fold, at least 2.5fold, at least 2.55 fold, at least 3.0 fold, at least 3.5 fold, at least4.0 fold, at least 4.5 fold, at least 5.0 fold, at least 5.5 fold, atleast 6.0 fold, at least 6.5 fold, at least 7.0 fold, at least 7.5 fold,at least 8.0 fold, at least 8.5 fold, at least 9.0 fold, at least 9.5fold, or at least 10 fold, for example, as compared to rate ofosteoclastogenesis in a corresponding subject that is not treated withthe agonist anti-TREM2 and/or anti-DAP12 antibody.

As used herein, the rate of osteoclastogenesis may be decreased if therate of osteoclastogenesis in a subject treated with an antagonistanti-TREM2 antibody and/or anti-DAP12 antibody of the present disclosureis smaller than the rate of osteoclastogenesis in a correspondingsubject that is not treated with the antagonist anti-TREM2 antibodyand/or anti-DAP12 antibody. In some embodiments, an antagonistanti-TREM2 antibody and/or anti-DAP12 antibody of the present disclosuremay decrease the rate of osteoclastogenesis in a subject by at least10%, at least 15%, at least 20%, at least 25%, at least 30%, at least35%, at least 40%, at least 45%, at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, at least 100%, at least 110%, at least115%, at least 120%, at least 125%, at least 130%, at least 135%, atleast 140%, at least 145%, at least 150%, at least 160%, at least 170%,at least 180%, at least 190%, or at least 200% for example, as comparedto rate of osteoclastogenesis in a corresponding subject that is nottreated with the antagonist anti-TREM2 antibody and/or anti-DAP12antibody. In other embodiments, an antagonist anti-TREM2 antibody and/oranti-DAP12 antibody of the present disclosure may decrease the rate ofosteoclastogenesis in a subject by at least 1.5 fold, at least 1.6 fold,at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2.0fold, at least 2.1 fold, at least 2.15 fold, at least 2.2 fold, at least2.25 fold, at least 2.3 fold, at least 2.35 fold, at least 2.4 fold, atleast 2.45 fold, at least 2.5 fold, at least 2.55 fold, at least 3.0fold, at least 3.5 fold, at least 4.0 fold, at least 4.5 fold, at least5.0 fold, at least 5.5 fold, at least 6.0 fold, at least 6.5 fold, atleast 7.0 fold, at least 7.5 fold, at least 8.0 fold, at least 8.5 fold,at least 9.0 fold, at least 9.5 fold, or at least 10 fold, for example,as compared to rate of osteoclastogenesis in a corresponding subjectthat is not treated with the antagonist anti-TREM2 antibody and/oranti-DAP12 antibody.

Without wishing to be bound by theory, it is believed that anti-TREM2and/or anti-DAP12 antibodies of the present disclosure are beneficialfor preventing, lowering the risk of, or treating conditions and/ordiseases associated with a reduction in osteoclast production and/or therate of osteoclastogenesis, including dementia, frontotemporal dementia,Alzheimer's disease, Nasu-Hakola disease, Parkinson's disease,Amyotrophic lateral sclerosis, Huntington's disease, Taupathy disease,and/or multiple sclerosis; or associated with abnormal bone formationand maintenance including osteoporosis, which is associated withpathological decrease in bone density and osteoporotic diseases whichare associated with pathological increase in bone density.

Proliferation and Survival of Macrophages, Microglial Cells, DendriticCells Monocytes, Osteoclasts, Langerhans Cells of Skin, and KupfferCells

In some embodiments, the anti-TREM2 and/or anti-DAP12 antibodies of thepresent disclosure may increase the proliferation, survival, and/orfunction of macrophages, microglial cells (microglia), dendritic cellsmonocytes, osteoclasts, Langerhans cells of skin, and Kupffer cellsafter binding to TREM2 and/or DAP12 protein expressed in a cell.

Microglial cells are a type of glial cell that are the residentmacrophages of the brain and spinal cord, and thus act as the first andmain form of active immune defense in the central nervous system (CNS).Microglial cells constitute 20% of the total glial cell populationwithin the brain. Microglial cells are constantly scavenging the CNS forplaques, damaged neurons and infectious agents. The brain and spinalcord are considered “immune privileged” organs in that they areseparated from the rest of the body by a series of endothelial cellsknown as the blood-brain barrier, which prevents most infections fromreaching the vulnerable nervous tissue. In the case where infectiousagents are directly introduced to the brain or cross the blood-brainbarrier, microglial cells must react quickly to decrease inflammationand destroy the infectious agents before they damage the sensitiveneural tissue. Due to the unavailability of antibodies from the rest ofthe body (few antibodies are small enough to cross the blood brainbarrier), microglia must be able to recognize foreign bodies, swallowthem, and act as antigen-presenting cells activating T-cells. Since thisprocess must be done quickly to prevent potentially fatal damage,microglial cells are extremely sensitive to even small pathologicalchanges in the CNS. They achieve this sensitivity in part by havingunique potassium channels that respond to even small changes inextracellular potassium.

As used herein, macrophages of the present disclosure include, withoutlimitation, M1 macrophages, activated M1 macrophages, and M2macrophages. As used herein, microglial cells of the present disclosureinclude, without limitation, M1 microglial cells, activated M1microglial cells, and M2 microglial 1 cells.

In some embodiments, anti-TREM2 and/or anti-DAP12 antibodies of thepresent disclosure may increase the expression of CD83 and/or CD86 ondendritic cells monocytes, and/or macrophages.

As used herein, the rate of proliferation, survival, and/or function ofmacrophages microglia, monocytes, and/or dendritic cells may includeincreased expression if the rate of proliferation, survival, and/orfunction of macrophages, microglia, dendritic cells monocytes,osteoclasts, Langerhans cells of skin, and/or Kupffer cells in a subjecttreated with an anti-TREM2 and/or anti-DAP12 antibody of the presentdisclosure is greater than the rate of proliferation, survival, and/orfunction of macrophages, microglia, dendritic cells monocytes,osteoclasts, Langerhans cells of skin, and/or Kupffer cells in acorresponding subject that is not treated with the anti-TREM2 and/oranti-DAP12 antibody. In some embodiments, an anti-TREM2 and/oranti-DAP12 antibody of the present disclosure may increase the rate ofproliferation, survival, and/or function of macrophages, microglia,dendritic cells monocytes, osteoclasts, Langerhans cells of skin, and/orKupffer cells in a subject by at least 10%, at least 15%, at least 20%,at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 100%, at least 110%, at least 115%, at least 120%, at least 125%,at least 130%, at least 135%, at least 140%, at least 145%, at least150%, at least 160%, at least 170%, at least 180%, at least 190%, or atleast 200% for example, as compared to the rate of proliferation,survival, and/or function of macrophages, microglia, dendritic cellsmonocytes, osteoclasts, Langerhans cells of skin, and/or Kupffer cellsin a corresponding subject that is not treated with the anti-TREM2and/or anti-DAP12 antibody. In other embodiments, an anti-TREM2 and/oranti-DAP12 antibody of the present disclosure may increase the rate ofproliferation, survival, and/or function of macrophages, microglia,dendritic cells monocytes, osteoclasts, Langerhans cells of skin, and/orKupffer cells in a subject by at least 1.5 fold, at least 1.6 fold, atleast 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2.0 fold,at least 2.1 fold, at least 2.15 fold, at least 2.2 fold, at least 2.25fold, at least 2.3 fold, at least 2.35 fold, at least 2.4 fold, at least2.45 fold, at least 2.5 fold, at least 2.55 fold, at least 3.0 fold, atleast 3.5 fold, at least 4.0 fold, at least 4.5 fold, at least 5.0 fold,at least 5.5 fold, at least 6.0 fold, at least 6.5 fold, at least 7.0fold, at least 7.5 fold, at least 8.0 fold, at least 8.5 fold, at least9.0 fold, at least 9.5 fold, or at least 10 fold, for example, ascompared to the rate of proliferation, survival, and/or function ofmacrophages, microglia, dendritic cells monocytes, osteoclasts,Langerhans cells of skin, and/or Kupffer cells in a correspondingsubject that is not treated with the anti-TREM2 and/or anti-DAP12antibody.

Without wishing to be bound by theory, it is believed that anti-TREM2and/or anti-DAP12 antibodies of the present disclosure are beneficialfor preventing, lowering the risk of, or treating conditions and/ordiseases associated with a reduction in proliferation, survival, and/orfunction of macrophages, microglia, dendritic cells monocytes,osteoclasts, Langerhans cells of skin, and/or Kupffer cells, includingdementia, frontotemporal dementia, Alzheimer's disease, Nasu-Hakoladisease, Parkinson's disease, Amyotrophic lateral sclerosis,Huntington's disease, Taupathy disease, and/or multiple sclerosis.

Clearance and Phagocytosis

In some embodiments, the anti-TREM2 and/or anti-DAP12 antibodies of thepresent disclosure may induce clearance and/or phagocytosis afterbinding to a TREM2 and/or DAP12 protein expressed in a cell of one ormore of apoptotic neurons, nerve tissue debris of the nervous system,non-nerve tissue debris of the nervous system, bacteria, other foreignbodies, disease-causing proteins, disease-causing peptides, and/ordisease-causing nucleic acids. In certain embodiments, disease-causingprotein include, without limitation, amyloid beta or fragments thereof,Tau, IAPP, alpha-synuclein, TDP-43, FUS protein, prion protein, PrPSc,huntingtin, calcitonin, superoxide dismutase, ataxin, Lewy body, atrialnatriuretic factor, islet amyloid polypeptide, insulin, apolipoproteinAI, serum amyloid A, medin, prolactin, transthyretin, lysozyme, beta 2microglobulin, gelsolin, keratoepithelin, cystatin, immunoglobulin lightchain AL, S-IBM protein, Repeat-associated non-ATG (RAN) translationproducts, DiPeptide repeat (DPR) peptides, glycine-alanine (GA) repeatpeptides, glycine-proline (GP) repeat peptides, glycine-arginine (GR)repeat peptides, proline-alanine (PA) repeat peptides, andproline-arginine (PR) repeat peptides. In certain embodiments,disease-causing nucleic acids include, without limitation, antisenseGGCCCC (G2C4) repeat-expansion RNA.

In some embodiments, the anti-TREM2 and/or anti-DAP12 antibodies of thepresent disclosure may induce of one or more types of clearance,including without limitation, apoptotic neuron clearance, nerve tissuedebris clearance, non-nerve tissue debris clearance, bacteria or otherforeign body clearance, disease-causing protein clearance,disease-causing peptide clearance, disease-causing nucleic acidclearance, and tumor cell clearance.

In some embodiments, the anti-TREM2 and/or anti-DAP12 antibodies of thepresent disclosure may induce phagocytosis of one or more of apoptoticneurons, nerve tissue debris, non-nerve tissue debris, bacteria, otherforeign bodies, disease-causing proteins, disease-causing peptides,disease-causing nucleic acid, and/or tumor cells.

In some embodiments, the anti-TREM2 and/or anti-DAP12 antibodies of thepresent disclosure may increase phagocytosis by macrophages, dendriticcells, monocytes, and/or microglia under conditions of reduced levels ofmacrophage colony-stimulating factor (MCSF). Alternatively, in someembodiments, the anti-TREM2 and/or anti-DAP12 antibodies of the presentdisclosure may decrease phagocytosis by macrophages, dendritic cells,monocytes, and/or microglia in the presence of normal levels ofmacrophage colony-stimulating factor (MCSF).

Without wishing to be bound by theory, it is believed that anti-TREM2and/or anti-DAP12 antibodies of the present disclosure are beneficialfor preventing, lowering the risk of, or treating conditions and/ordiseases associated with apoptotic neurons, nerve tissue debris of thenervous system, non-nerve tissue debris of the nervous system, bacteria,other foreign bodies, or disease-causing proteins, including dementia,frontotemporal dementia, Alzheimer's disease, Nasu-Hakola disease,Parkinson's disease, Amyotrophic lateral sclerosis, Huntington'sdisease, Taupathy disease, and/or multiple sclerosis.

Syk Phosphorylation

In some embodiments, the anti-TREM2 and/or anti-DAP12 antibodies of thepresent disclosure may induce spleen tyrosine kinase (Syk)phosphorylation after binding to a TREM2 and/or DAP12 protein expressedin a cell.

Spleen tyrosine kinase (Syk) is an intracellular signaling molecule thatfunctions downstream of TREM2 by phosphorylating several substrates,thereby facilitating the formation of a signaling complex leading tocellular activation and inflammatory processes.

Without wishing to be bound by theory, it is believed that anti-TREM2and/or anti-DAP12 antibodies of the present disclosure are beneficialfor preventing, lowering the risk of, or treating conditions and/ordiseases associated with decreased levels of Syk phosphorylation,including dementia, frontotemporal dementia, Alzheimer's disease,Nasu-Hakola disease, Parkinson's disease, Amyotrophic lateral sclerosis,Huntington's disease, Taupathy disease, and/or multiple sclerosis.

TREM2-Dependent Gene Expression

In some embodiments, agonist anti-TREM2 and/or anti-DAP12 antibodies ofthe present disclosure may increase the activity and/or expression ofTREM2-dependent genes, such as one or more transcription factors of thenuclear factor of activated T-cells (NFAT) family of transcriptionfactors. Alternatively, antagonistic anti-TREM2 and/or anti-DAP12antibodies of the present disclosure may inhibit the activity and/orexpression of TREM2-dependent genes, such as one or more transcriptionfactors of the NFAT family of transcription factors.

Without wishing to be bound by theory, it is believed that agonistanti-TREM2 and/or anti-DAP12 antibodies of the present disclosure arebeneficial for preventing, lowering the risk of, or treating conditionsand/or diseases associated with decreased levels of TREM2-dependentgenes, including dementia, frontotemporal dementia, Alzheimer's disease,Nasu-Hakola disease, Parkinson's disease, Amyotrophic lateral sclerosis,Huntington's disease, Taupathy disease, and/or multiple sclerosis.

Antibody Preparation

Anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure canencompass polyclonal antibodies, monoclonal antibodies, humanized andchimeric antibodies, human antibodies, antibody fragments (e.g., Fab,Fab′-SH, Fv, scFv, and F(ab′)₂), bispecific and polyspecific antibodies,multivalent antibodies, library derived antibodies, antibodies havingmodified effector functions, fusion proteins containing an antibodyportion, and any other modified configuration of the immunoglobulinmolecule that includes an antigen recognition site, such as an epitopehaving amino acid residues of a TREM2 and/or DAP12 protein of thepresent disclosure, including glycosylation variants of antibodies,amino acid sequence variants of antibodies, and covalently modifiedantibodies. The anti-TREM2 and/or anti-DAP12 antibodies may be human,murine, rat, or of any other origin (including chimeric or humanizedantibodies).

(1) Polyclonal Antibodies

Polyclonal antibodies, such as anti-TREM2 and/or anti-DAP12 polyclonalantibodies, are generally raised in animals by multiple subcutaneous(sc) or intraperitoneal (ip) injections of the relevant antigen and anadjuvant. It may be useful to conjugate the relevant antigen (e.g., apurified or recombinant TREM2 and/or DAP12 protein of the presentdisclosure) to a protein that is immunogenic in the species to beimmunized, e.g., keyhole limpet hemocyanin (KLH), serum albumin, bovinethyroglobulin, or soybean trypsin inhibitor, using a bifunctional orderivatizing agent, e.g., maleimidobenzoyl sulfosuccinimide ester(conjugation through cysteine residues), N-hydroxysuccinimide (throughlysine residues), glutaraldehyde, succinic anhydride, SOCl₂, orR¹N═C═NR, where R and R¹ are independently lower alkyl groups. Examplesof adjuvants which may be employed include Freund's complete adjuvantand MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalosedicorynomycolate). The immunization protocol may be selected by oneskilled in the art without undue experimentation.

The animals are immunized against the desired antigen, immunogenicconjugates, or derivatives by combining, e.g., 100 μg (for rabbits) or 5μg (for mice) of the protein or conjugate with 3 volumes of Freund'scomplete adjuvant and injecting the solution intradermally at multiplesites. One month later, the animals are boosted with ⅕ to 1/10 theoriginal amount of peptide or conjugate in Freund's complete adjuvant bysubcutaneous injection at multiple sites. Seven to fourteen days later,the animals are bled and the serum is assayed for antibody titer.Animals are boosted until the titer plateaus. Conjugates also can bemade in recombinant-cell culture as protein fusions. Also, aggregatingagents such as alum are suitable to enhance the immune response.

(2) Monoclonal Antibodies

Monoclonal antibodies, such as anti-TREM2 and/or anti-DAP12 monoclonalantibodies, are obtained from a population of substantially homogeneousantibodies, i.e., the individual antibodies comprising the populationare identical except for possible naturally occurring mutations and/orpost-translational modifications (e.g., isomerizations, amidations) thatmay be present in minor amounts. Thus, the modifier “monoclonal”indicates the character of the antibody as not being a mixture ofdiscrete antibodies.

For example, the anti-TREM2 and/or anti-DAP12 monoclonal antibodies maybe made using the hybridoma method first described by Köhler et al.,Nature, 256:495 (1975), or may be made by recombinant DNA methods (U.S.Pat. No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster, is immunized as hereinabove described to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the protein used for immunization (e.g., apurified or recombinant TREM2 and/or DAP12 protein of the presentdisclosure). Alternatively, lymphocytes may be immunized in vitro.Lymphocytes then are fused with myeloma cells using a suitable fusingagent, such as polyethylene glycol, to form a hybridoma cell (Goding,Monoclonal Antibodies: Principles and Practice, pp. 59-103 (AcademicPress, 1986)).

The immunizing agent will typically include the antigenic protein (e.g.,a purified or recombinant TREM2 and/or DAP12 protein of the presentdisclosure) or a fusion variant thereof. Generally peripheral bloodlymphocytes (“PBLs”) are used if cells of human origin are desired,while spleen or lymph node cells are used if non-human mammalian sourcesare desired. The lymphoctyes are then fused with an immortalized cellline using a suitable fusing agent, such as polyethylene glycol, to forma hybridoma cell. Goding, Monoclonal Antibodies: Principles andPractice, Academic Press (1986), pp. 59-103.

Immortalized cell lines are usually transformed mammalian cells,particularly myeloma cells of rodent, bovine or human origin. Usually,rat or mouse myeloma cell lines are employed. The hybridoma cells thusprepared are seeded and grown in a suitable culture medium thatpreferably contains one or more substances that inhibit the growth orsurvival of the unfused, parental myeloma cells. For example, if theparental myeloma cells lack the enzyme hypoxanthine guaninephosphoribosyl transferase (HGPRT or HPRT), the culture medium for thehybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which are substances that prevent the growth ofHGPRT-deficient-cells.

Preferred immortalized myeloma cells are those that fuse efficiently,support stable high-level production of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. Among these, preferred are murine mycloma lines, such as thosederived from MOPC-21 and MPC-11 mouse tumors (available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA), as well asSP-2 cells and derivatives thereof (e.g., X63-Ag8-653) (available fromthe American Type Culture Collection, Manassas, Va. USA). Human myelomaand mouse-human heteromyeloma cell lines have also been described forthe production of human monoclonal antibodies (Kozbor, J. Immunol.,133:3001 (1984); Brodeur et al., Monoclonal Antibody ProductionTechniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York,1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen (e.g.,a TREM2 and/or DAP12 protein of the present disclosure). Preferably, thebinding specificity of monoclonal antibodies produced by hybridoma cellsis determined by immunoprecipitation or by an in vitro binding assay,such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay(ELISA).

The culture medium in which the hybridoma cells are cultured can beassayed for the presence of monoclonal antibodies directed against thedesired antigen (e.g., a TREM2 and/or DAP12 protein of the presentdisclosure). Preferably, the binding affinity and specificity of themonoclonal antibody can be determined by immunoprecipitation or by an invitro binding assay, such as radioimmunoassay (RIA) or enzyme-linkedassay (ELISA). Such techniques and assays are known in the in art. Forexample, binding affinity may be determined by the Scatchard analysis ofMunson et al., Anal. Biochem., 107:220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, supra). Suitable culture media for this purpose include, forexample, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells maybe grown in vivo as tumors in a mammal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose chromatography, hydroxylapatitechromatography, gel electrophoresis, dialysis, affinity chromatography,and other methods as described above.

Anti-TREM2 and/or anti-DAP12 monoclonal antibodies may also be made byrecombinant DNA methods, such as those disclosed in U.S. Pat. No.4,816,567, and as described above. DNA encoding the monoclonalantibodies is readily isolated and sequenced using conventionalprocedures (e.g., by using oligonucleotide probes that specifically bindto genes encoding the heavy and light chains of murine antibodies). Thehybridoma cells serve as a preferred source of such DNA. Once isolated,the DNA may be placed into expression vectors, which are thentransfected into host-cells such as E. coli cells, simian COS cells,Chinese hamster ovary (CHO) cells, or myeloma cells that do nototherwise produce immunoglobulin protein, in order to synthesizemonoclonal antibodies in such recombinant host-cells. Review articles onrecombinant expression in bacteria of DNA encoding the antibody includeSkerra et al., Curr. Opin. Immunol., 5:256-262 (1993) and Plückthun,Immunol. Rev. 130:151-188 (1992).

In certain embodiments, anti-TREM2 and/or anti-DAP12 antibodies can beisolated from antibody phage libraries generated using the techniquesdescribed in McCafferty et al., Nature, 348:552-554 (1990). Clackson etal., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol.,222:581-597 (1991) described the isolation of murine and humanantibodies, respectively, from phage libraries. Subsequent publicationsdescribe the production of high affinity (nanomolar (“nM”) range) humanantibodies by chain shuffling (Marks et al., Bio/Technology, 10:779-783(1992)), as well as combinatorial infection and in vivo recombination asa strategy for constructing very large phage libraries (Waterhouse etal., Nucl. Acids Res., 21:2265-2266 (1993)). Thus, these techniques areviable alternatives to traditional monoclonal antibody hybridomatechniques for isolation of monoclonal antibodies of desired specificity(e.g., those that bind a TREM2 protein of the present disclosure).

The DNA encoding antibodies or fragments thereof may also be modified,for example, by substituting the coding sequence for human heavy- andlight-chain constant domains in place of the homologous murine sequences(U.S. Pat. No. 4,816,567; Morrison, et al., Proc. Natl Acad. Sci. USA,81:6851 (1984)), or by covalently joining to the immunoglobulin codingsequence all or part of the coding sequence for a non-immunoglobulinpolypeptide. Typically such non-immunoglobulin polypeptides aresubstituted for the constant domains of an antibody, or they aresubstituted for the variable domains of one antigen-combining site of anantibody to create a chimeric bivalent antibody comprising oneantigen-combining site having specificity for an antigen and anotherantigen-combining site having specificity for a different antigen.

The monoclonal antibodies described herein (e.g., anti-TREM2 and/oranti-DAP12 antibodies of the present disclosure or fragments thereof)may by monovalent, the preparation of which is well known in the art.For example, one method involves recombinant expression ofimmunoglobulin light chain and a modified heavy chain. The heavy chainis truncated generally at any point in the Fc region so as to preventheavy chain crosslinking. Alternatively, the relevant cysteine residuesmay be substituted with another amino acid residue or are deleted so asto prevent crosslinking. In vitro methods are also suitable forpreparing monovalent antibodies. Digestion of antibodies to producefragments thereof, particularly Fab fragments, can be accomplished usingroutine techniques known in the art.

Chimeric or hybrid anti-TREM2 and/or anti-DAP12 antibodies also may beprepared in vitro using known methods in synthetic protein chemistry,including those involving crosslinking agents. For example, immunotoxinsmay be constructed using a disulfide-exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate.

(3) Humanized Antibodies

Anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure orantibody fragments thereof may further include humanized or humanantibodies. Humanized forms of non-human (e.g., murine) antibodies arechimeric immunoglobulins, immunoglobulin chains or fragments thereof(such as Fab, Fab′-SH, Fv, scFv, F(ab′)₂ or other antigen-bindingsubsequences of antibodies) which contain minimal sequence derived fromnon-human immunoglobulin. Humanized antibodies include humanimmunoglobulins (recipient antibody) in which residues from acomplementarity determining region (CDR) of the recipient are replacedby residues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity andcapacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally will also comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. Jones et al., Nature 321: 522-525 (1986); Riechmann etal., Nature 332: 323-329 (1988) and Presta, Curr. Opin. Struct. Biol. 2:593-596 (1992).

Methods for humanizing non-human anti-TREM2 and/or anti-DAP12 antibodiesare well known in the art. Generally, a humanized antibody has one ormore amino acid residues introduced into it from a source which isnon-human. These non-human amino acid residues are often referred to as“import” residues, which are typically taken from an “import” variabledomain. Humanization can be essentially performed following the methodof Winter and co-workers, Jones et al., Nature 321:522-525 (1986);Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science239:1534-1536 (1988), or through substituting rodent CDRs or CDRsequences for the corresponding sequences of a human antibody.Accordingly, such “humanized” antibodies are chimeric antibodies (U.S.Pat. No. 4,816,567), wherein substantially less than an intact humanvariable domain has been substituted by the corresponding sequence froma non-human species. In practice, humanized antibodies are typicallyhuman antibodies in which some CDR residues and possibly some FRresidues are substituted by residues from analogous sites in rodentantibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework (FR) for the humanized antibody. Sims et al., J.Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901(1987). Another method uses a particular framework derived from theconsensus sequence of all human antibodies of a particular subgroup oflight or heavy chains. The same framework may be used for severaldifferent humanized antibodies. Carter et al., Proc. Nat'l Acad. Sci.USA 89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993).

Furthermore, it is important that antibodies be humanized with retentionof high affinity for the antigen and other favorable biologicalproperties. To achieve this goal, according to a preferred method,humanized antibodies are prepared by a process of analyzing the parentalsequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen or antigens (e.g., TREM2 proteins of the presentdisclosure), is achieved. In general, the CDR residues are directly andmost substantially involved in influencing antigen binding.

Various forms of the humanized anti-TREM2 and/or anti-DAP12 antibody arecontemplated. For example, the humanized anti-TREM2 and/or anti-DAP12antibody may be an antibody fragment, such as an Fab, which isoptionally conjugated with one or more TREM2 ligand, such as HSP60.Alternatively, the humanized anti-TREM2 and/or anti-DAP12 antibody maybe an intact antibody, such as an intact IgG1 antibody.

(4) Human Antibodies

Alternatively, human anti-TREM2 and/or anti-DAP12 antibodies can begenerated. For example, it is now possible to produce transgenic animals(e.g., mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. The homozygous deletion of the antibodyheavy-chain joining region (J_(H)) gene in chimeric and germ-line mutantmice results in complete inhibition of endogenous antibody production.Transfer of the human germ-line immunoglobulin gene array in suchgerm-line mutant mice will result in the production of human antibodiesupon antigen challenge. See, e.g., Jakobovits et al., Proc. Nat'l Acad.Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993);Bruggermann et al., Year in Immunol., 7:33 (1993); U.S. Pat. No.5,591,669 and WO 97/17852.

Alternatively, phage display technology can be used to produce humananti-TREM2 and/or anti-DAP12 antibodies and antibody fragments in vitro,from immunoglobulin variable (V) domain gene repertoires fromunimmunized donors. McCafferty et al., Nature 348:552-553 (1990);Hoogenboom and Winter, J. Mol. Biol. 227: 381 (1991). According to thistechnique, antibody V domain genes are cloned in-frame into either amajor or minor coat protein gene of a filamentous bacteriophage, such asM13 or fd, and displayed as functional antibody fragments on the surfaceof the phage particle. Because the filamentous particle contains asingle-stranded DNA copy of the phage genome, selections based on thefunctional properties of the antibody also result in selection of thegene encoding the antibody exhibiting those properties. Thus, the phagemimics some of the properties of the B-cell. Phage display can beperformed in a variety of formats, reviewed in, e.g., Johnson, Kevin S.and Chiswell, David J., Curr. Opin Struct. Biol. 3:564-571 (1993).Several sources of V-gene segments can be used for phage display.Clackson et al., Nature 352:624-628 (1991) isolated a diverse array ofanti-oxazolone antibodies from a small random combinatorial library of Vgenes derived from the spleens of immunized mice. A repertoire of Vgenes from unimmunized human donors can be constructed and antibodies toa diverse array of antigens (including self-antigens) can be isolatedessentially following the techniques described by Marks et al., J. Mol.Biol. 222:581-597 (1991), or Griffith et al., EMBO J. 12:725-734 (1993).See also U.S. Pat. Nos. 5,565,332 and 5,573,905. Additionally, yeastdisplay technology can be used to produce human anti-TREM2 and/oranti-DAP12 antibodies and antibody fragments in vitro (e.g., WO2009/036379; WO 2010/105256; WO 2012/009568; US 2009/0181855; US2010/0056386; and Feldhaus and Siegel (2004) J. Immunological Methods290:69-80). In other embodiments, ribosome display technology can beused to produce human anti-TREM2 and/or anti-DAP12 antibodies andantibody fragments in vitro (e.g., Roberts and Szostak (1997) Proc NatlAcad Sci 94:12297-12302; Schaffitzel et al. (1999) J. ImmunolicalMethods 231:119-135; Lipovsek and Plückthun (2004) J. ImmunologicalMethods 290:51-67).

The techniques of Cole et al., and Boerner et al., are also availablefor the preparation of human anti-TREM2 and/or anti-DAP12 monoclonalantibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, AlanR. Liss, p. 77 (1985) and Boerner et al., J. Immunol. 147(1): 86-95(1991). Similarly, human anti-TREM2 and/or anti-DAP12 antibodies can bemade by introducing human immunoglobulin loci into transgenic animals,e.g., mice in which the endogenous immunoglobulin genes have beenpartially or completely inactivated. Upon challenge, human antibodyproduction is observed, which closely resembles that seen in humans inall respects, including gene rearrangement, assembly and antibodyrepertoire. This approach is described, for example, in U.S. Pat. Nos.5,545,807; 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,661,016 and inthe following scientific publications: Marks et al., Bio/Technology 10:779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison,Nature 368: 812-13 (1994), Fishwild et al., Nature Biotechnology 14:845-51 (1996), Neuberger, Nature Biotechnology 14: 826 (1996) andLonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995).

Finally, human anti-TREM2 and/or anti-DAP12 antibodies may also begenerated in vitro by activated B-cells (see U.S. Pat. Nos. 5,567,610and 5,229,275).

(5) Antibody Fragments

In certain embodiments there are advantages to using anti-TREM2 and/oranti-DAP12 antibody fragments, rather than whole anti-TREM2 and/oranti-DAP12 antibodies. In some embodiments, smaller fragment sizes allowfor rapid clearance and better brain penetration.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., J. Biochem.Biophys. Method. 24:107-117 (1992); and Brennan et al., Science 229:81(1985)). However, these fragments can now be produced directly byrecombinant host-cells, for example, using nucleic acids encodinganti-TREM2 and/or anti-DAP12 antibodies of the present disclosure. Fab,Fv and scFv antibody fragments can all be expressed in and secreted fromE. coli, thus allowing the straightforward production of large amountsof these fragments. Anti-TREM2 and/or anti-DAP12 antibody fragments canalso be isolated from the antibody phage libraries as discussed above.Alternatively, Fab′-SH fragments can be directly recovered from E. coliand chemically coupled to form F(ab′)₂ fragments (Carter et al.,Bio/Technology 10:163-167 (1992)). According to another approach,F(ab′)₂ fragments can be isolated directly from recombinant host-cellculture. Production of Fab and F(ab′)₂ antibody fragments with increasedin vivo half-lives are described in U.S. Pat. No. 5,869,046. In otherembodiments, the antibody of choice is a single chain Fv fragment(scFv). See WO 93/16185; U.S. Pat. No. 5,571,894 and U.S. Pat. No.5,587,458. The anti-TREM2 and/or anti-DAP12 antibody fragment may alsobe a “linear antibody,” e.g., as described in U.S. Pat. No. 5,641,870.Such linear antibody fragments may be monospecific or bispecific.

(6) Bispecific and Polyspecific Antibodies

Bispecific antibodies (BsAbs) are antibodies that have bindingspecificities for at least two different epitopes, including those onthe same or another protein (e.g., one or more TREM2 proteins of thepresent disclosure). Alternatively, one part of a BsAb can be armed tobind to the target TREM2 and/or DAP12 antigen, and another can becombined with an arm that binds to a second protein. Such antibodies canbe derived from full-length antibodies or antibody fragments (e.g.,F(ab′)₂ bispecific antibodies).

Methods for making bispecific antibodies are known in the art.Traditional production of full-length bispecific antibodies is based onthe coexpression of two immunoglobulin heavy-chain/light chain pairs,where the two chains have different specificities. Millstein et al.,Nature, 305:537-539 (1983). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829 and in Traunecker et al., EMBOJ., 10:3655-3659 (1991).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion preferablyis with an immunoglobulin heavy chain constant domain, comprising atleast part of the hinge, C_(H)2, and C_(H)3 regions. It is preferred tohave the first heavy-chain constant region (C_(H)1) containing the sitenecessary for light chain binding, present in at least one of thefusions. DNAs encoding the immunoglobulin heavy chain fusions and, ifdesired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable hostorganism. This provides for great flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yields. It is, however, possible to insert thecoding sequences for two or all three polypeptide chains in oneexpression vector when the expression of at least two polypeptide chainsin equal ratios results in high yields or when the ratios are of noparticular significance.

In a preferred embodiment of this approach, the bispecific antibodiesare composed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyhalf of the bispecific molecules provides for an easy way of separation.This approach is disclosed in WO 94/04690. For further details ofgenerating bispecific antibodies, see, for example, Suresh et al.,Methods in Enzymology 121: 210 (1986); and Garber, Nature Reviews DrugDiscovery 13, 799-801 (2014).

According to another approach described in WO 96/27011 or U.S. Pat. No.5,731,168, the interface between a pair of antibody molecules can beengineered to maximize the percentage of heterodimers which arerecovered from recombinant-cell culture. The preferred interfacecomprises at least a part of the C_(H)3 region of an antibody constantdomain. In this method, one or more small amino acid side chains fromthe interface of the first antibody molecule are replaced with largerside chains (e.g., tyrosine or tryptophan). Compensatory “cavities” ofidentical or similar size to the large side chains(s) are created on theinterface of the second antibody molecule by replacing large amino acidside chains with smaller ones (e.g., alanine or threonine). Thisprovides a mechanism for increasing the yield of the heterodimer overother unwanted end-products such as homodimers.

Techniques for generating bispecific antibodies from antibody fragmentshave been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science 229:81 (1985) describe a procedure wherein intact antibodies areproteolytically cleaved to generate F(ab′)₂ fragments. These fragmentsare reduced in the presence of the dithiol complexing agent sodiumarsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-TNB derivative to form the bispecificantibody. The bispecific antibodies produced can be used as agents forthe selective immobilization of enzymes.

Fab′ fragments may be directly recovered from E. coli and chemicallycoupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175:217-225 (1992) describes the production of fully humanized bispecificantibody F(ab′)₂ molecules. Each Fab′ fragment was separately secretedfrom E. coli and subjected to directed chemical coupling in vitro toform the bispecific antibody. The bispecific antibody thus formed wasable to bind to cells overexpressing the ErbB2 receptor and normal humanT-cells, as well as trigger the lytic activity of human cytotoxiclymphocytes against human breast tumor targets.

Various techniques for making and isolating bivalent antibody fragmentsdirectly from recombinant-cell culture have also been described. Forexample, bivalent heterodimers have been produced using leucine zippers.Kostelny et al., J. Immunol., 148(5):1547-1553 (1992). The leucinezipper peptides from the Fos and Jun proteins were linked to the Fab′portions of two different antibodies by gene fusion. The antibodyhomodimers were reduced at the hinge region to form monomers and thenre-oxidized to form the antibody heterodimers. The “diabody” technologydescribed by Hollinger et al., Proc. Nat'l Acad. Sci. USA, 90: 6444-6448(1993) has provided an alternative mechanism for makingbispecific/bivalent antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific/bivalentantibody fragments by the use of single-chain Fv (sFv) dimers has alsobeen reported. See Gruber et al., J. Immunol., 152:5368 (1994).

Another method to generate bispecific antibodies is controlled Fab-armexchange (cFAE), which is an easy-to-use method to generate bispecificIgG1 (bsIgG1). The protocol involves the following: (i) separateexpression of two parental IgG1s containing single matching pointmutations in the CH3 domain; (ii) mixing of parental IgG1 s underpermissive redox conditions in vitro to enable recombination ofhalf-molecules; (iii) removal of the reductant to allow reoxidation ofinterchain disulfide bonds; and (iv) analysis of exchange efficiency andfinal product using chromatography-based or mass spectrometry (MS)-basedmethods. The protocol generates bsAbs with regular IgG architecture,characteristics and quality attributes both at bench scale (microgramsto milligrams) and at a mini-bioreactor scale (milligrams to grams) thatis designed to model large-scale manufacturing (kilograms). Startingfrom good-quality purified proteins, exchange efficiencies of ≥95% canbe obtained within 2-3 days (including quality control). See Labrijn etal., Natur Protocols 9, 2450-2463 (2014); and Garber, Nature ReviewsDrug Discovery 13, 799-801 (2014).

Antibodies with more than two valencies are also contemplated. Forexample, trispecific antibodies can be prepared. Tutt et al., J.Immunol. 147:60 (1991).

Exemplary bispecific antibodies may bind to two different epitopes on agiven molecule (e.g., a TREM2 and/or DAP12 protein of the presentdisclosure). In some embodiments a bispecific antibody binds to a firstantigen, such as a TREM2 or DAP12 protein of the present disclosure, anda second antigen facilitating transport across the blood-brain barrier.Numerous antigens are known in the art that facilitate transport acrossthe blood-brain barrier (see, e.g., Gabathuler R., Approaches totransport therapeutic drugs across the blood-brain barrier to treatbrain diseases, Neurobiol. Dis. 37 (2010) 48-57). Such second antigensinclude, without limitation, transferrin receptor (TR), insulin receptor(HIR), Insulin-like growth factor receptor (IGFR), low-densitylipoprotein receptor related proteins 1 and 2 (LPR-1 and 2), diphtheriatoxin receptor, including CRM197 (a non-toxic mutant of diphtheriatoxin), llama single domain antibodies such as TMEM 30(A) (Flippase),protein transduction domains such as TAT, Syn-B, or penetratin,poly-arginine or generally positively charged peptides, Angiopeppeptides such as ANG1005 (see, e.g., Gabathuler, 2010), and other cellsurface proteins that are enriched on blood-brain barrier endothelialcells (see, e.g., Daneman et al., PLoS One. 2010 Oct. 29; 5(10):e13741).In some embodiments, second antigens for an anti-TREM2 antibody mayinclude, without limitation, a DAP12 antigen of the present disclosure.In other embodiments, second antigens for an anti-DAP12 antibody mayinclude, without limitation, a TREM2 antigen of the present disclosure.In other embodiments, bispecific antibodies that bind to both TREM2 andDAP12 may facilitate and enhance one or more TREM2 and/or DAP12activities. In other embodiments, second antigens for an TREM2 and/orDAP12 antibody may include, without limitation, A beta peptide, antigenor an alpha synuclain protein antigene or, Tau protein antigene or,TDP-43 protein antigene or, prion protein antigene or, huntingtinprotein antigene, or RAN, translation Products antigene, including theDiPeptide Repeats, (DPRs peptides) composed of glycine-alanine (GA),glycine-proline (GP), glycine-arginine (GR), proline-alanine (PA), orproline-arginine (PR).

(7) Multivalent Antibodies

A multivalent antibody may be internalized (and/or catabolized) fasterthan a bivalent antibody by a cell expressing an antigen to which theantibodies bind. The anti-TREM2 and/or anti-DAP12 antibodies of thepresent disclosure or antibody fragments thereof can be multivalentantibodies (which are other than of the IgM class) with three or moreantigen binding sites (e.g., tetravalent antibodies), which can bereadily produced by recombinant expression of nucleic acid encoding thepolypeptide chains of the antibody. The multivalent antibody cancomprise a dimerization domain and three or more antigen binding sites.The preferred dimerization domain comprises an Fc region or a hingeregion. In this scenario, the antibody will comprise an Fc region andthree or more antigen binding sites amino-terminal to the Fc region. Thepreferred multivalent antibody herein contains three to about eight, butpreferably four, antigen binding sites. The multivalent antibodycontains at least one polypeptide chain (and preferably two polypeptidechains), wherein the polypeptide chain or chains comprise two or morevariable domains. For instance, the polypeptide chain or chains maycomprise VD1-(X1)n-VD2-(X2)n-Fc, wherein VD1 is a first variable domain,VD2 is a second variable domain, Fc is one polypeptide chain of an Fcregion, X1 and X2 represent an amino acid or polypeptide, and n is 0or 1. Similarly, the polypeptide chain or chains may compriseV_(H)-C_(H)1-flexible linker-V_(H)-C_(H)1-Fc region chain; orV_(H)-C_(H)1-V_(H)-C_(H)1-Fc region chain. The multivalent antibodyherein preferably further comprises at least two (and preferably four)light chain variable domain polypeptides. The multivalent antibodyherein may, for instance, comprise from about two to about eight lightchain variable domain polypeptides. The light chain variable domainpolypeptides contemplated here comprise a light chain variable domainand, optionally, further comprise a CL domain. The multivalentantibodies may recognize a TREM2 angigen as well as without limitationadditional antigens A beta peptide, antigen or an alpha synuclainprotein antigene or, Tau protein antigene or, TDP-43 protein antigeneor, prion protein antigene or, huntingtin protein antigene, or RAN,translation Products antigene, including the DiPeptide Repeats, (DPRspeptides) composed of glycine-alanine (GA), glycine-proline (GP),glycine-arginine (GR), proline-alanine (PA), or proline-arginine (PR),Isnulin receptor, insulin like growth factor receptor. Transferrinreceptor or any other antigen that facilitate antibody transfer acrossthe blood brain barrier.

(8) Effector Function Engineering

It may also be desirable to modify an anti-TREM2 and/or anti-DAP12antibody of the present disclosure to modify effector function and/or toincrease serum half-life of the antibody. For example, the Fc receptorbinding site on the constant region may be modified or mutated to removeor reduce binding affinity to certain Fc receptors, such as FcγRI,FcγRII, and/or FcγRIII (e.g., to reduce antibody-dependent cell-mediatedcytotoxicity. In some embodiments, the effector function is impaired byremoving N-glycosylation of the Fc region (e.g., in the CH 2 domain ofIgG) of the antibody. In some embodiments, the effector function isimpaired by modifying regions such as 233-236, 297, and/or 327-331 ofhuman IgG as described in PCT WO 99/58572 and Armour et al., MolecularImmunology 40: 585-593 (2003); Reddy et al., J. Immunology 164:1925-1933(2000). In some embodiments, it may also be desirable to modify ananti-TREM2 and/or anti-DAP12 antibody of the present disclosure tomodify effector function to increase selectivity toward theITIM-containing FcgRIIb (CD32b) to increase clustering of antibodies onadjacent cells without activating humoral responses includingantibody-dependent cell-mediated cytotoxicity and antibody-dependentcellular phagocytosis.

To increase the serum half-life of the antibody, one may incorporate asalvage receptor binding epitope into the antibody (especially anantibody fragment) as described in U.S. Pat. No. 5,739,277, for example.As used herein, the term “salvage receptor binding epitope” refers to anepitope of the Fc region of an IgG molecule (e.g., IgG₁, IgG₂, IgG₃, orIgG₄) that is responsible for increasing the in vivo serum half-life ofthe IgG molecule.

(9) Other Amino Acid Sequence Modifications

Amino acid sequence modifications of anti-TREM2 and/or anti-DAP12antibodies of the present disclosure, or antibody fragments thereof, arealso contemplated. For example, it may be desirable to improve thebinding affinity and/or other biological properties of the antibodies orantibody fragments. Amino acid sequence variants of the antibodies orantibody fragments are prepared by introducing appropriate nucleotidechanges into the nucleic acid encoding the antibodies or antibodyfragments, or by peptide synthesis. Such modifications include, forexample, deletions from, and/or insertions into and/or substitutions of,residues within the amino acid sequences of the antibody. Anycombination of deletion, insertion, and substitution is made to arriveat the final construct, provided that the final construct possesses thedesired characteristics (i.e., the ability to bind or physicallyinteract with a TREM2 and/or DAP12 protein of the present disclosure).The amino acid changes also may alter post-translational processes ofthe antibody, such as changing the number or position of glycosylationsites.

A useful method for identification of certain residues or regions of theanti-TREM2 and/or anti-DAP12 antibody that are preferred locations formutagenesis is called “alanine scanning mutagenesis” as described byCunningham and Wells in Science, 244:1081-1085 (1989). Here, a residueor group of target residues are identified (e.g., charged residues suchas arg, asp, his, lys, and glu) and replaced by a neutral or negativelycharged amino acid (most preferably alanine or polyalanine) to affectthe interaction of the amino acids with the target antigen. Those aminoacid locations demonstrating functional sensitivity to the substitutionsthen are refined by introducing further or other variants at, or for,the sites of substitution. Thus, while the site for introducing an aminoacid sequence variation is predetermined, the nature of the mutation perse need not be predetermined. For example, to analyze the performance ofa mutation at a given site, alanine scanning or random mutagenesis isconducted at the target codon or region and the expressed antibodyvariants are screened for the desired activity.

Amino acid sequence insertions include amino- (“N”) and/or carboxy-(“C”) terminal fusions ranging in length from one residue topolypeptides containing a hundred or more residues, as well asintrasequence insertions of single or multiple amino acid residues.Examples of terminal insertions include an antibody with an N-terminalmethionyl residue or the antibody fused to a cytotoxic polypeptide.Other insertional variants of the antibody molecule include the fusionto the N- or C-terminus of the antibody to an enzyme or a polypeptidewhich increases the serum half-life of the antibody.

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the antibody moleculereplaced by a different residue. The sites of greatest interest forsubstitutional mutagenesis include the hypervariable regions, but FRalterations are also contemplated. Conservative substitutions are shownin the Table A below under the heading of “preferred substitutions”. Ifsuch substitutions result in a change in biological activity, then moresubstantial changes, denominated “exemplary substitutions” in Table A,or as further described below in reference to amino acid classes, may beintroduced and the products screened.

TABLE A Amino Acid Substitutions Original Exemplary Preferred ResidueSubstitutions Substitutions Ala (A) val; leu; ile val Arg (R) lys; gln;asn lys Asn (N) gln; his; asp, lys; arg gln Asp (D) glu; asn glu Cys (C)ser; ala ser Gln (Q) asn; glu asn Glu (E) asp; gln asp Gly (G) ala alaHis (H) asn; gln; lys; arg arg Ile (I) leu; val; met; ala; phe;norleucine leu Leu (L) norleucine; ile; val; met; ala; phe ile Lys (K)arg; gln; asn arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; ala;tyr tyr Pro (P) ala ala Ser (S) thr thr Thr (T) Ser ser Trp (W) tyr; phetyr Tyr (Y) trp; phc; thr; scr phc Val (V) ile; leu; met; phe; ala;norleucine leu

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Naturallyoccurring residues are divided into groups based on common side-chainproperties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gln, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe.

Non-conservative substitutions entail exchanging a member of one ofthese classes for another class.

Any cysteine residue not involved in maintaining the proper conformationof the antibody also may be substituted, generally with serine, toimprove the oxidative stability of the molecule and prevent aberrantcrosslinking. Conversely, cysteine bond(s) may be added to the antibodyto improve its stability (particularly where the antibody is an antibodyfragment, such as an Fv fragment).

A particularly preferred type of substitutional variant involvessubstituting one or more hypervariable region residues of a parentantibody (e.g. a humanized or human anti-TREM2 and/or anti-DAP12antibody). Generally, the resulting variant(s) selected for furtherdevelopment will have improved biological properties relative to theparent antibody from which they are generated. A convenient way forgenerating such substitutional variants involves affinity maturationusing phage display. Briefly, several hypervariable region sites (e.g.,6-7 sites) are mutated to generate all possible amino substitutions ateach site. The antibody variants thus generated are displayed in amonovalent fashion from filamentous phage particles as fusions to thegene III product of M13 packaged within each particle. Thephage-displayed variants are then screened for their biological activity(e.g., binding affinity) as herein disclosed. In order to identifycandidate hypervariable region sites for modification, alanine scanningmutagenesis can be performed to identify hypervariable region residuescontributing significantly to antigen binding. Alternatively, oradditionally, it may be beneficial to analyze a crystal structure of theantigen-antibody complex to identify contact points between the antibodyand the antigen (e.g., a TREM2 protein of the present disclosure). Suchcontact residues and neighboring residues are candidates forsubstitution according to the techniques elaborated herein. Once suchvariants are generated, the panel of variants is subjected to screeningas described herein and antibodies with superior properties in one ormore relevant assays may be selected for further development.

Another type of amino acid variant of the antibody alters the originalglycosylation pattern of the antibody. By altering is meant deleting oneor more carbohydrate moieties found in the antibody, and/or adding oneor more glycosylation sites that are not present in the antibody.

Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly scrine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).

Nucleic acid molecules encoding amino acid sequence variants of theanti-IgE antibody are prepared by a variety of methods known in the art.These methods include, but are not limited to, isolation from a naturalsource (in the case of naturally occurring amino acid sequence variants)or preparation by oligonucleotide-mediated (or site-directed)mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlierprepared variant or a non-variant version of the antibodies (e.g.,anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure) orantibody fragments.

(10) Other Antibody Modifications

Anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure, orantibody fragments thereof, can be further modified to containadditional non-proteinaceous moieties that are known in the art andreadily available, or to contain different types of drug conjugates thatare known in the art and readily available. Preferably, the moietiessuitable for derivatization of the antibody are water-soluble polymers.Non-limiting examples of water-soluble polymers include, but are notlimited to, polyethylene glycol (PEG), copolymers of ethyleneglycol/propylene glycol, carboxymethylcellulose, dextran, polyvinylalcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, polypropylene glycol homopolymers,polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols(e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethyleneglycol propionaldehyde may have advantages in manufacturing due to itsstability in water. The polymer may be of any molecular weight, and maybe branched or unbranched. The number of polymers attached to theantibody may vary, and if more than one polymer is attached, they can bethe same or different molecules. In general, the number and/or type ofpolymers used for derivatization can be determined based onconsiderations including, but not limited to, the particular propertiesor functions of the antibody to be improved, whether the antibodyderivative will be used in a therapy under defined conditions, etc. Suchtechniques and other suitable formulations are disclosed in Remington:The Science and Practice of Pharmacy, 20th Ed., Alfonso Gennaro, Ed.,Philadelphia College of Pharmacy and Science (2000).

Drug conjugation involves coupling of a biological active cytotoxic(anticancer) payload or drug to an antibody that specifically targets acertain tumor marker (e.g. a protein that, ideally, is only to be foundin or on tumor cells). Antibodies track these proteins down in the bodyand attach themselves to the surface of cancer cells. The biochemicalreaction between the antibody and the target protein (antigen) triggersa signal in the tumor cell, which then absorbs or internalizes theantibody together with the cytotoxin. After the ADC is internalized, thecytotoxic drug is released and kills the cancer. Due to this targeting,ideally the drug has lower side effects and gives a wider therapeuticwindow than other chemotherapeutic agents. Technics to conjugateantibodies are disclosed are known in the art (see, e.g., Jane deLartigue, OncLive Jul. 5, 2012; ADC Review on antibody-drug conjugates;and Ducry et al., (2010). Bioconjugate Chemistry 21 (1): 5-13).

Binding Assays and Other Assays

Anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure (e.g.,antibody activity of anti-TREM2 antibodies) may be identified, screenedfor, and/or characterized for their physical/chemical properties and/orbiological activities by various assays known in the art or assaysdescribed in the Examples herein, such as, for example, radiolabeledimmunoassays, optical assays, protein binding assays, biochemicalscreening assays, immunoassays, fluorescence assays, and/or cellsurvival assays.

In some embodiments, anti-TREM2 and/or anti-DAP12 antibodies of thepresent disclosure may be tested for antigen binding activity, e.g., byknown methods such as ELISA, Western blot, etc.

In some embodiments, competition assays may be used to identify anantibody that competes with any of the antibodies listed in Tableland/or Table 8, selected from Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8,Ab9, Ab10, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19, Ab20,Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33,Ab34, Ab35, Ab36, Ab37, Ab38, Ab39, Ab40, Ab41, Ab42, Ab43, Ab44, Ab45,Ab46, Ab47, Ab48, Ab49, Ab50, Ab51, Ab53, Ab54, Ab55, Ab56, Ab57, Ab58,Ab59, Ab60, Ab61, Ab62, Ab63, Ab64, Ab65, Ab66, Ab67, Ab68, Ab69, Ab70,Ab71, Ab72, Ab73, Ab74, Ab75, Ab76, Ab77, Ab78, Ab79, Ab80, Ab81, Ab82,Ab83, Ab84, Ab85, Ab86, and Ab87, and/or human and/or humanized MAB17291for binding to TREM2. In certain embodiments, such a competing antibodybinds to the same epitope (e.g., a linear or a conformational epitope)that is bound by any of the antibodies listed in Table land/or Table 8,selected from Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11,Ab12, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19, Ab20, Ab22, Ab23, Ab24,Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, Ab34, Ab35, Ab36,Ab37, Ab38, Ab39, Ab40, Ab41, Ab42, Ab43, Ab44, Ab45, Ab46, Ab47, Ab48,Ab49, Ab50, Ab51, Ab53, Ab54, Ab55, Ab56, Ab57, Ab58, Ab59, Ab60, Ab61,Ab62, Ab63, Ab64, Ab65, Ab66, Ab67, Ab68, Ab69, Ab70, Ab71, Ab72, Ab73,Ab74, Ab75, Ab76, Ab77, Ab78, Ab79, Ab80, Ab81, Ab82, Ab83, Ab84, Ab85,Ab86, and Ab87, and/or human and/or humanized MAB17291. Detailedexemplary methods for mapping an epitope to which an antibody binds areprovided in Morris (1996) “Epitope Mapping Protocols,” in Methods inMolecular Biology vol. 66 (Humana Press, Totowa, N.J.).

In an exemplary competition assay, immobilized TREM2 or cells expressingTREM2 on cell surface are incubated in a solution comprising a firstlabeled antibody that binds to TREM2 (e.g., human or non-human primate)and a second unlabeled antibody that is being tested for its ability tocompete with the first antibody for binding to TREM2. The secondantibody may be present in a hybridoma supernatant. As a control,immobilized TREM2 or cells expressing TREM2 is incubated in a solutioncomprising the first labeled antibody but not the second unlabeledantibody. After incubation under conditions permissive for binding ofthe first antibody to TREM2, excess unbound antibody is removed, and theamount of label associated with immobilized TREM2 or cells expressingTREM2 is measured. If the amount of label associated with immobilizedTREM2 or cells expressing TREM2 is substantially reduced in the testsample relative to the control sample, then that indicates that thesecond antibody is competing with the first antibody for binding toTREM2. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14(Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

In some embodiments, assays are provided for identifying agonistanti-TRME2 and/or anti-DAP12 antibodies of the present disclosure havingbiological activity. Biological activity may include, e.g., inducing,promoting, stimulating, or otherwise increasing one more activities ofTREM2 and/or DAP12. Agonist anti-TRME2 and/or anti-DAP12 antibodieantibodies having such biological activity in vivo and/or in vitro arealso provided.

Assays known in the art and described herein (see, e.g., Examples 23-27,33-38, 41-44, 52-55, and 67-68) can be used for identifying and testingbiological activities of anti-TRME2 and/or anti-DAP12 antibodies thatinduce, promote, stimulate, or otherwise increase one more activities ofTREM2 and/or DAP12. In some embodiments, assays for testing one or moreactivities of an agonist anti-TRME2 and/or anti-DAP12 antibody areprovided. An exemplary test for biological activity may include, e.g.,treating a population of immune cells, such as an innate immune cellsexpressing TREM2 and/or DAP12 with an agonist anti-TRME2 and/oranti-DAP12 antibody candidate for sufficient time to allow binding to,and activation of, the TREM2 and/or DAP12 protein, and measuring cellsurvival (or cell death) as compared to cell survival (or cell death) ina corresponding population in the absence of the antibody candidate.Methods for measuring cell survival or cell death are well known in theart and described herein. Agonist anti-TRME2 and/or anti-DAP12antibodies may be identified by their ability to promote or prolong cellsurvival or delay cell death in a cell population relative to a controlcell population, e.g., a cell population that was not treated with theagonist antibody candidate. Certain aspects of the present disclosureprovide methods for identifying a candidate agonist antibody thatspecifically binds to TREM2 and/or DAP12. In certain embodiments, themethod for identifying a candidate agonist antibody that specificallybinds to TREM2 and/or DAP12 comprises a. contacting an innate immunecell population expressing TREM2 and/or DAP12 on its surface with acandidate agonist antibody that specifically binds to the TREM2 and/orDAP12 for a period of time sufficient for the candidate agonist antibodyto promote cell survival in the innate immune cell population; and b.comparing cell survival of the contacted innate immune cell populationcontacted cell with cell survival from a corresponding innate immunecell population not contacted with the candidate agonist antibody thatspecifically binds to the TREM2 and/or DAP12, wherein increased cellsurvival from the contacted innate immune cell population indicates thatthe candidate antibody is an agonist. Other aspects of the presentdisclosure provide an isolated antibody that specifically binds to TREM2and/or DAP12 identified by any of the disclosed methods for identifyingan antibody that specifically binds to TREM2 and/or DAP12.

Nucleic Acids, Vectors, and Host Cells

Anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure may beproduced using recombinant methods and compositions, e.g., as describedin U.S. Pat. No. 4,816,567. In some embodiments, isolated nucleic acidshaving a nucleotide sequence encoding any of the anti-TREM2 and/oranti-DAP12 antibodies of the present disclosure are provided. Suchnucleic acids may encode an amino acid sequence containing the VL and/oran amino acid sequence containing the VH of the anti-TREM2 and/oranti-DAP12 antibody (e.g., the light and/or heavy chains of theantibody). In some embodiments, one or more vectors (e.g., expressionvectors) containing such nucleic acids are provided. In someembodiments, a host cell containing such nucleic acid is also provided.In some embodiments, the host cell is an isolated host cell. As usedherein, an “isolated cell” is a a cell that is identified and separatedfrom at least one contaminant cell with which it is ordinarilyassociated in the environment in which it was produced. In someembodiments, the isolated cell is free of association with allcomponents associated with the production environment. The isolated cellis in a form other than in the form or setting in which it is found innature. Isolated cells are distinguished from cells existing naturallyin tissues, organs, or individuals. In some embodiments, the isolatedcell is a host cell of the present disclosure. In some embodiments, thehost cell contains (e.g., has been transduced with): (1) a vectorcontaining a nucleic acid that encodes an amino acid sequence containingthe VL of the antibody and an amino acid sequence containing the VH ofthe antibody, or (2) a first vector containing a nucleic acid thatencodes an amino acid sequence containing the VL of the antibody and asecond vector containing a nucleic acid that encodes an amino acidsequence containing the VH of the antibody. In some embodiments, thehost cell is eukaryotic, e.g., a Chinese Hamster Ovary (CHO) cell orlymphoid cell (e.g., Y0, NS0, Sp20 cell). Host cells of the presentdisclosure also include, without limitation, isolated cells, in vitrocultured cells, and ex vivo cultured cells.

Methods of making an anti-TREM2 and/or anti-DAP12 antibody of thepresent disclosure are provided. In some embodiments, the methodincludes culturing a host cell of the present disclosure containing anucleic acid encoding the anti-TREM2 and/or anti-DAP12 antibody, underconditions suitable for expression of the antibody. In some embodiments,the antibody is subsequently recovered from the host cell (or host cellculture medium).

For recombinant production of an anti-TREM2 and/or anti-DAP12 antibodyof the present disclosure, a nucleic acid encoding the anti-TREM2 and/oranti-DAP12 antibody is isolated and inserted into one or more vectorsfor further cloning and/or expression in a host cell. Such nucleic acidmay be readily isolated and sequenced using conventional procedures(e.g., by using oligonucleotide probes that are capable of bindingspecifically to genes encoding the heavy and light chains of theantibody).

Suitable vectors containing a nucleic acid sequence encoding any of theanti-TREM2 and/or anti-DAP12 antibodies of the present disclosure, orfragments thereof polypeptides (including antibodies) described hereininclude, without limitation, cloning vectors and expression vectors.Suitable cloning vectors can be constructed according to standardtechniques, or may be selected from a large number of cloning vectorsavailable in the art. While the cloning vector selected may varyaccording to the host cell intended to be used, useful cloning vectorsgenerally have the ability to self-replicate, may possess a singletarget for a particular restriction endonuclease, and/or may carry genesfor a marker that can be used in selecting clones containing the vector.Suitable examples include plasmids and bacterial viruses, e.g., pUC18,pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mpl8, mpl9,pBR322, pMB9, ColE1, pCR1, RP4, phage DNAs, and shuttle vectors such aspSA3 and pAT28. These and many other cloning vectors are available fromcommercial vendors such as BioRad, Strategene, and Invitrogen.

Expression vectors generally are replicable polynucleotide constructsthat contain a nucleic acid of the present disclosure. The expressionvector may replicable in the host cells either as episomes or as anintegral part of the chromosomal DNA. Suitable expression vectorsinclude but are not limited to plasmids, viral vectors, includingadenoviruses, adeno-associated viruses, retroviruses, cosmids, andexpression vector(s) disclosed in PCT Publication No. WO 87/04462.Vector components may generally include, but are not limited to, one ormore of the following: a signal sequence; an origin of replication; oneor more marker genes; suitable transcriptional controlling elements(such as promoters, enhancers and terminator). For expression (i.e.,translation), one or more translational controlling elements are alsousually required, such as ribosome binding sites, translation initiationsites, and stop codons.

The vectors containing the nucleic acids of interest can be introducedinto the host cell by any of a number of appropriate means, includingelectroporation, transfection employing calcium chloride, rubidiumchloride, calcium phosphate, DEAE-dextran, or other substances;microprojectile bombardment; lipofection; and infection (e.g., where thevector is an infectious agent such as vaccinia virus). The choice ofintroducing vectors or polynucleotides will often depend on features ofthe host cell. In some embodiments, the vector contains a nucleic acidcontaining one or more amino acid sequences encoding an anti-TREM2and/or anti-DAP12 antibody of the present disclosure.

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells. For example, anti-TREM2and/or anti-DAP12 antibodies of the present disclosure may be producedin bacteria, in particular when glycosylation and Fc effector functionare not needed. For expression of antibody fragments and polypeptides inbacteria (e.g., U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523; andCharlton, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed.,Humana Press, Totowa, N.J., 2003), pp. 245-254, describing expression ofantibody fragments in E. coli.). After expression, the antibody may beisolated from the bacterial cell paste in a soluble fraction and can befurther purified.

In addition to prokaryotes, eukaryotic microorganisms, such asfilamentous fungi or yeast, are also suitable cloning or expressionhosts for antibody-encoding vectors, including fungi and yeast strainswhose glycosylation pathways have been “humanized,” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern (e.g., Gerngross, Nat. Biotech. 22:1409-1414 (2004); and Li etal., Nat. Biotech. 24:210-215 (2006)).

Suitable host cells for the expression of glycosylated antibody can alsobe derived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains have been identified which may be used inconjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells. Plant cell cultures can also be utilized ashosts (e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978,and 6,417,429, describing PLANTIBODIES™ technology for producingantibodies in transgenic plants.).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977));baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells asdescribed, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkeykidney cells (CV1); African green monkey kidney cells (VERO-76); humancervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo ratliver cells (BRL 3A); human lung cells (W138); human liver cells (HepG2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., inMather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; andFS4 cells. Other useful mammalian host cell lines include Chinesehamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al.,Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines suchas Y0, NS0 and Sp2/0. For a review of certain mammalian host cell linessuitable for antibody production, see, e.g., Yazaki and Wu, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,N.J.), pp. 255-268 (2003).

Pharmaceutical Compositions

Anti-TREM2 and/or anti-DAP12 antibodies of the present disclosure can beincorporated into a variety of formulations for therapeuticadministration by combining the antibodies with appropriatepharmaceutically acceptable carriers or diluents, and may be formulatedinto preparations in solid, semi-solid, liquid or gaseous forms.Examples of such formulations include, without limitation, tablets,capsules, powders, granules, ointments, solutions, suppositories,injections, inhalants, gels, microspheres, and aerosols. Pharmaceuticalcompositions can include, depending on the formulation desired,pharmaceutically-acceptable, non-toxic carriers of diluents, which arevehicles commonly used to formulate pharmaceutical compositions foranimal or human administration. The diluent is selected so as not toaffect the biological activity of the combination. Examples of suchdiluents include, without limitation, distilled water, buffered water,physiological saline, PBS, Ringer's solution, dextrose solution, andHank's solution. A pharmaceutical composition or formulation of thepresent disclosure can further include other carriers, adjuvants, ornon-toxic, nontherapeutic, nonimmunogenic stabilizers, excipients andthe like. The compositions can also include additional substances toapproximate physiological conditions, such as pH adjusting and bufferingagents, toxicity adjusting agents, wetting agents and detergents.

A pharmaceutical composition of the present disclosure can also includeany of a variety of stabilizing agents, such as an antioxidant forexample. When the pharmaceutical composition includes a polypeptide, thepolypeptide can be complexed with various well-known compounds thatenhance the in vivo stability of the polypeptide, or otherwise enhanceits pharmacological properties (e.g., increase the half-life of thepolypeptide, reduce its toxicity, and enhance solubility or uptake).Examples of such modifications or complexing agents include, withoutlimitation, sulfate, gluconate, citrate and phosphate. The polypeptidesof a composition can also be complexed with molecules that enhance theirin vivo attributes. Such molecules include, without limitation,carbohydrates, polyamines, amino acids, other peptides, ions (e.g.,sodium, potassium, calcium, magnesium, manganese), and lipids.

Further examples of formulations that are suitable for various types ofadministration can be found in Remington's Pharmaceutical Sciences, MacePublishing Company, Philadelphia, Pa., 17th ed. (1985). For a briefreview of methods for drug delivery, see, Langer, Science 249:1527-1533(1990).

For oral administration, the active ingredient can be administered insolid dosage forms, such as capsules, tablets, and powders, or in liquiddosage forms, such as elixirs, syrups, and suspensions. The activecomponent(s) can be encapsulated in gelatin capsules together withinactive ingredients and powdered carriers, such as glucose, lactose,sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesiumstearate, stearic acid, sodium saccharin, talcum, magnesium carbonate.Examples of additional inactive ingredients that may be added to providedesirable color, taste, stability, buffering capacity, dispersion orother known desirable features are red iron oxide, silica gel, sodiumlauryl sulfate, titanium dioxide, and edible white ink. Similar diluentscan be used to make compressed tablets. Both tablets and capsules can bemanufactured as sustained release products to provide for continuousrelease of medication over a period of hours. Compressed tablets can besugar coated or film coated to mask any unpleasant taste and protect thetablet from the atmosphere, or enteric-coated for selectivedisintegration in the gastrointestinal tract. Liquid dosage forms fororal administration can contain coloring and flavoring to increasepatient acceptance.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.

The components used to formulate the pharmaceutical compositions arepreferably of high purity and are substantially free of potentiallyharmful contaminants (e.g., at least National Food (NF) grade, generallyat least analytical grade, and more typically at least pharmaceuticalgrade). Moreover, compositions intended for in vivo use are usuallysterile. To the extent that a given compound must be synthesized priorto use, the resulting product is typically substantially free of anypotentially toxic agents, particularly any endotoxins, which may bepresent during the synthesis or purification process. Compositions forparental administration are also sterile, substantially isotonic andmade under GMP conditions.

Formulations may be optimized for retention and stabilization in thebrain or central nervous system. When the agent is administered into thecranial compartment, it is desirable for the agent to be retained in thecompartment, and not to diffuse or otherwise cross the blood brainbarrier. Stabilization techniques include cross-linking, multimerizing,or linking to groups such as polyethylene glycol, polyacrylamide,neutral protein carriers, etc. in order to achieve an increase inmolecular weight.

Other strategies for increasing retention include the entrapment of theantibody, such as an anti-TREM2 and/or anti-DAP12 antibody of thepresent disclosure, in a biodegradable or bioerodible implant. The rateof release of the therapeutically active agent is controlled by the rateof transport through the polymeric matrix, and the biodegradation of theimplant. The transport of drug through the polymer barrier will also beaffected by compound solubility, polymer hydrophilicity, extent ofpolymer cross-linking, expansion of the polymer upon water absorption soas to make the polymer barrier more permeable to the drug, geometry ofthe implant, and the like. The implants are of dimensions commensuratewith the size and shape of the region selected as the site ofimplantation. Implants may be particles, sheets, patches, plaques,fibers, microcapsules and the like and may be of any size or shapecompatible with the selected site of insertion.

The implants may be monolithic, i.e. having the active agenthomogenously distributed through the polymeric matrix, or encapsulated,where a reservoir of active agent is encapsulated by the polymericmatrix. The selection of the polymeric composition to be employed willvary with the site of administration, the desired period of treatment,patient tolerance, the nature of the disease to be treated and the like.Characteristics of the polymers will include biodegradability at thesite of implantation, compatibility with the agent of interest, ease ofencapsulation, a half-life in the physiological environment.

Biodegradable polymeric compositions that may be employed include,without limitation, organic esters or ethers, which when degraded resultin physiologically acceptable degradation products, including themonomers. Anhydrides, amides, orthoesters or the like, by themselves orin combination with other monomers, may find use. The polymers will becondensation polymers. The polymers may be cross-linked ornon-cross-linked. Of particular interest are polymers ofhydroxyaliphatic carboxylic acids, either homo- or copolymers, andpolysaccharides. Included among the polyesters of interest are polymersof D-lactic acid, L-lactic acid, racemic lactic acid, glycolic acid,polycaprolactone, and combinations thereof. By employing the L-lactateor D-lactate, a slowly biodegrading polymer is achieved, whiledegradation is substantially enhanced with the racemate. Copolymers ofglycolic and lactic acid are of particular interest, where the rate ofbiodegradation is controlled by the ratio of glycolic to lactic acid.The most rapidly degraded copolymer has roughly equal amounts ofglycolic and lactic acid, where either homopolymer is more resistant todegradation. The ratio of glycolic acid to lactic acid will also affectthe brittleness of in the implant, where a more flexible implant isdesirable for larger geometries. Among the polysaccharides of interestare calcium alginate, and functionalized celluloses, particularlycarboxymethylcellulose esters characterized by being water insoluble, amolecular weight of about 5 kD to 500 kD, etc. Biodegradable hydrogelsmay also be employed in the implants of the subject invention. Hydrogelsare typically a copolymer material, characterized by the ability toimbibe a liquid. Exemplary biodegradable hydrogels which may be employedare described in Heller in: Hydrogels in Medicine and Pharmacy, N. A.Peppes ed., Vol. III, CRC Press, Boca Raton, Fla., 1987, pp 137-149.

Pharmaceutical Dosages

Pharmaceutical compositions of the present disclosure containing ananti-TREM2 and/or anti-DAP12 antibody of the present disclosure may beadministered to an individual in need of treatment with the anti-TREM2and/or anti-DAP12 antibody, preferably a human, in accord with knownmethods, such as intravenous administration as a bolus or by continuousinfusion over a period of time, by intramuscular, intraperitoneal,intracerobrospinal, intracranial, intraspinal, subcutaneous,intra-articular, intrasynovial, intrathecal, oral, topical, orinhalation routes.

Dosages and desired drug concentration of pharmaceutical compositions ofthe present disclosure may vary depending on the particular useenvisioned. The determination of the appropriate dosage or route ofadministration is well within the skill of an ordinary artisan. Animalexperiments provide reliable guidance for the determination of effectivedoses for human therapy. Interspecies scaling of effective doses can beperformed following the principles described in Mordenti, J. andChappell, W. “The Use of Interspecies Scaling in Toxicokinetics,” InToxicokinetics and New Drug Development, Yacobi et al., Eds, PergamonPress, New York 1989, pp.42-46.

For in vivo administration of any of the anti-TREM2 and/or anti-DAP12antibodies of the present disclosure, normal dosage amounts may varyfrom about 10 ng/kg up to about 100 mg/kg of an individual's body weightor more per day, preferably about 1 mg/kg/day to 10 mg/kg/day, dependingupon the route of administration. For repeated administrations overseveral days or longer, depending on the severity of the disease,disorder, or condition to be treated, the treatment is sustained until adesired suppression of symptoms is achieved.

An exemplary dosing regimen may include administering an initial dose ofan anti-TREM2 and/or anti-DAP12 antibody, of about 2 mg/kg, followed bya weekly maintenance dose of about 1 mg/kg every other week. Otherdosage regimens may be useful, depending on the pattern ofpharmacokinetic decay that the physician wishes to achieve. For example,dosing an individual from one to twenty-one times a week is contemplatedherein. In certain embodiments, dosing ranging from about 3 μg/kg toabout 2 mg/kg (such as about 3 μg/kg, about 10 μg/kg, about 30 μg/kg,about 100 μg/kg, about 300 μg/kg, about 1 mg/kg, and about 2/mg/kg) maybe used. In certain embodiments, dosing frequency is three times perday, twice per day, once per day, once every other day, once weekly,once every two weeks, once every four weeks, once every five weeks, onceevery six weeks, once every seven weeks, once every eight weeks, onceevery nine weeks, once every ten weeks, or once monthly, once every twomonths, once every three months, or longer. Progress of the therapy iseasily monitored by conventional techniques and assays. The dosingregimen, including the anti-TREM2 and/or anti-DAP12 antibodyadministered, can vary over time independently of the dose used.

Dosages for a particular anti-TREM2 and/or anti-DAP12 antibody may bedetermined empirically in individuals who have been given one or moreadministrations of the anti-TREM2 and/or anti-DAP12 antibody.Individuals are given incremental doses of an anti-TREM2 and/oranti-DAP12 antibody. To assess efficacy of an anti-TREM2 and/oranti-DAP12 antibody, a clinical symptom of any of the diseases,disorders, or conditions of the present disclosure (e.g., dementia,frontotemporal dementia, Alzheimer's disease, Nasu-Hakola disease, andmultiple sclerosis) can be monitored.

Administration of an anti-TREM2 and/or anti-DAP12 antibody of thepresent disclosure can be continuous or intermittent, depending, forexample, on the recipient's physiological condition, whether the purposeof the administration is therapeutic or prophylactic, and other factorsknown to skilled practitioners. The administration of an anti-TREM2and/or anti-DAP12 antibody may be essentially continuous over apreselected period of time or may be in a series of spaced doses.

Guidance regarding particular dosages and methods of delivery isprovided in the literature; see, for example, U.S. Pat. Nos. 4,657,760;5,206,344; or 5,225,212. It is within the scope of the invention thatdifferent formulations will be effective for different treatments anddifferent disorders, and that administration intended to treat aspecific organ or tissue may necessitate delivery in a manner differentfrom that to another organ or tissue. Moreover, dosages may beadministered by one or more separate administrations, or by continuousinfusion. For repeated administrations over several days or longer,depending on the condition, the treatment is sustained until a desiredsuppression of disease symptoms occurs. However, other dosage regimensmay be useful. The progress of this therapy is easily monitored byconventional techniques and assays.

Therapeutic Uses

Further aspects of the present disclosure provide methods for modulating(e.g., activating or inhibiting) TREM2, modulating (e.g., activating orinhibiting) DAP12, modulating (e.g., activating or inhibiting) PI3K,modulating (e.g., increasing or reducing) expression of one or moreanti-inflammatory mediators (e.g., IL-12p70, IL-6, and IL-10), ormodulating (e.g., increasing or reducing) expression of one or morepro-inflammatory mediators (e.g., L-1β and TNF) in an individual in needthereof, by administering to the individual a therapeutically effectiveamount of an anti-TREM2 and/or anti-DAP12 antibody of the presentdisclosure to modulate (e.g., induce or inhibit) one or more TREM2and/or DAP12 activities in the individual.

As disclosed herein, anti-TREM2 and/or anti-DAP12 antibodies of thepresent disclosure may be used for preventing, reducing risk, ortreating dementia, frontotemporal dementia, Alzheimer's disease,vascular dementia, mixed dementia, Creutzfeldt-Jakob disease, normalpressure hydrocephalus, amyotrophic lateral sclerosis, Huntington'sdisease, Taupathy disease, Nasu-Hakola disease, stroke, acute trauma,chronic trauma, lupus, acute and chronic colitis, wound healing, Crohn'sdisease, inflammatory bowel disease, ulcerative colitis, obesity,Malaria, essential tremor, central nervous system lupus, Behcet'sdisease, Parkinson's disease, dementia with Lewy bodies, multiple systematrophy, Shy-Drager syndrome, progressive supranuclear palsy, corticalbasal ganglionic degeneration, acute disseminated encephalomyelitis,granulomartous disorders, Sarcoidosis, diseases of aging, seizures,spinal cord injury, traumatic brain injury, age related maculardegeneration, glaucoma, retinitis pigmentosa, retinal degeneration,respiratory tract infection, sepsis, eye infection, systemic infection,lupus, arthritis, multiple sclerosis, low bone density, osteoporosis,osteogenesis, osteopetrotic disease, Paget's disease of bone, and/or,cancer (e.g., bladder cancer breast cancer, colon and rectal cancer,endometrial cancer, kidney cancer, renal cell cancer, renal pelviscancer, leukemia, lung cancer, melanoma, non-Hodgkin's lymphoma,pancreatic cancer, prostate cancer, ovarian cancer, fibrosarcoma, andthyroid cancer). In some embodiments, the anti-TREM2 and/or anti-DAP12antibodies are agonist antibodies. In some embodiments, the anti-TREM2and/or anti-DAP12 antibodies are inert antibodies. In some embodiments,the anti-TREM2 and/or anti-DAP12 antibodies are antagonist antibodies.

In some embodiments, the present disclosure provides methods ofpreventing, reducing risk, or treating an individual having dementia,frontotemporal dementia, Alzheimer's disease, vascular dementia, mixeddementia, Creutzfeldt-Jakob disease, normal pressure hydrocephalus,amyotrophic lateral sclerosis, Huntington's disease, Taupathy disease,Nasu-Hakola disease, stroke, acute trauma, chronic trauma, lupus, acuteand chronic colitis, wound healing, Crohn's disease, inflammatory boweldisease, ulcerative colitis, obesity, Malaria, essential tremor, centralnervous system lupus, Behcet's disease, Parkinson's disease, dementiawith Lewy bodies, multiple system atrophy, Shy-Drager syndrome,progressive supranuclear palsy, cortical basal ganglionic degeneration,acute disseminated encephalomyelitis, granulomartous disorders,Sarcoidosis, diseases of aging, seizures, spinal cord injury, traumaticbrain injury, age related macular degeneration, glaucoma, retinitispigmentosa, retinal degeneration, respiratory tract infection, sepsis,eye infection, systemic infection, lupus, arthritis, multiple sclerosis,low bone density, osteoporosis, osteogenesis, osteopetrotic disease,Paget's disease of bone, and cancer, by administering to the individuala therapeutically effective amount of an anti-TREM2 and/or anti-DAP12antibody of the present disclosure to modulate (e.g., induce or inhibit)one or more TREM2 activities, including without limitation, DAP12phosphorylation, PI3K activation, expression of one or moreanti-inflammatory mediators (e.g., IL-12p70, IL-6, and IL-10), orexpression of one or more pro-inflammatory mediators (e.g., L-1β andTNF). In some embodiments, the anti-TREM2 and/or anti-DAP12 antibody isan agonist antibody. In some embodiments, the anti-TREM2 and/oranti-DAP12 antibody is an inert antibody. In some embodiments, theanti-TREM2 and/or anti-DAP12 antibody is an antagonist antibody. Incertain embodiments, the individual has a heterozygous TREM2 variantallele having an glutamic acid to stop codon substitution in the nucleicacid sequence encoding amino acid residue 14 of the human TREM2 protein(SEQ ID NO: 1). In certain embodiments, the individual has aheterozygous TREM2 variant allele having a glutamine to stop codonsubstitution in the nucleic acid sequence encoding amino acid residue 33of the human TREM2 protein (SEQ ID NO: 1). In certain embodiments, theindividual has a heterozygous TREM2 variant allele having a tryptophanto stop codon substitution in the nucleic acid sequence encoding aminoacid residue 44 of the human TREM2 protein (SEQ ID NO: 1). In certainembodiments, the individual has a heterozygous TREM2 variant allelehaving an arginine to histidine amino acid substitution at amino acidresidue 47 of the human TREM2 protein (SEQ ID NO: 1). In certainembodiments, the individual has a heterozygous TREM2 variant allelehaving a tryptophan to stop codon substitution in the nucleic acidsequence encoding amino acid residue 78 of the human TREM2 protein (SEQID NO: 1). In certain embodiments, the individual has a heterozygousTREM2 variant allele having a valine to glycine amino acid substitutionat an amino acid corresponding to amino acid residue 126 of the humanTREM2 protein (SEQ ID NO: 1). In certain embodiments, the individual hasa heterozygous TREM2 variant allele having an aspartic acid to glycineamino acid substitution at an amino acid corresponding to amino acidresidue 134 of the human TREM2 protein (SEQ ID NO: 1). In certainembodiments, the individual has a heterozygous TREM2 variant allelehaving a lysine to asparagine amino acid substitution at an amino acidcorresponding to amino acid residue 186 of the human TREM2 protein (SEQID NO: 1).

In some embodiments, the individual has a heterozygous TREM2 variantallele having a guanine nucleotide deletion at a nucleotidecorresponding to nucleotide residue G313 of the nucleic acid sequenceencoding SEQ ID NO: 1; a guanine nucleotide deletion at a nucleotidecorresponding to nucleotide residue G267 of the nucleic acid sequenceencoding SEQ ID NO: 1; a threonine to methionine amino acid substitutionat an amino acid corresponding to amino acid residue Thr66 of SEQ ID NO:1; and/or a serine to cysteine amino acid substitution at an amino acidcorresponding to amino acid residue Ser116 of SEQ ID NO: 1.

In some embodiments, the individual has a heterozygous DAP12 variantallele having a methionine to threonine substitution at an amino acidcorresponding to amino acid residue Met1 of SEQ ID NO: 2, a glycine toarginine amino acid substitution at an amino acid corresponding to aminoacid residue Gly49 of SEQ ID NO: 2, a deletion within exons 1-4 of thenucleic acid sequence encoding SEQ ID NO: 2, an insertion of 14 aminoacid residues at exon 3 of the nucleic acid sequence encoding SEQ ID NO:2, and/or a guanine nucleotide deletion at a nucleotide corresponding tonucleotide residue G141 of the nucleic acid sequence encoding SEQ ID NO:2.

As disclosed herein, anti-TREM2 and/or anti-DAP12 antibodies of thepresent disclosure may also be used for inducing and/or promoting innateimmune cell survival. In some embodiments, the present disclosureprovides methods of inducing or promoting innate immune cell survival inan individual in need thereof, by administering to the individual atherapeutically effective amount of an agonist anti-TREM2 and/oranti-DAP12 antibody of the present disclosure.

As disclosed herein, anti-TREM2 and/or anti-DAP12 antibodies of thepresent disclosure may also be used for inducing and/or promoting woundhealing, such as after injury. In some embodiments, the wound healingmay be colonic wound repair following injury. In some embodiments, thepresent disclosure provides methods of inducing or promoting woundhealing in an individual in need thereof, by administering to theindividual a therapeutically effective amount of an agonist anti-TREM2and/or anti-DAP12 antibody of the present disclosure.

As disclosed herein, anti-TREM2 and/or anti-DAP12 antibodies of thepresent disclosure may also be used for inhibiting innate immune cellsurvival. In some embodiments, the present disclosure provides methodsof i inhibiting innate immune cell survival in an individual in needthereof, by administering to the individual a therapeutically effectiveamount of an antagonist anti-TREM2 and/or anti-DAP12 antibody of thepresent disclosure.

In some embodiments, the methods of the present disclosure may involvethe coadministration of anti-TREM2 and/or anti-DAP12 antibodies, orbispecific antibodies that bind to both TREM2 and DAP12, with TLRantagonists or with agents neutralizing TLR agonist (e.g., neutralizingcytokine or interleukin antibodies).

In some embodiments, the methods of the present disclosure may involvethe administration of chimeric constructs, including an anti-TREM2and/or anti-DAP12 antibody of the present disclosure in conjunction witha TREM2 ligand, such as HSP60.

Dementia

Dementia is a non-specific syndrome (i.e., a set of signs and symptoms)that presents as a serious loss of global cognitive ability in apreviously unimpaired person, beyond what might be expected from normalageing. Dementia may be static as the result of a unique global braininjury. Alternatively, dementia may be progressive, resulting inlong-term decline due to damage or disease in the body. While dementiais much more common in the geriatric population, it can also occurbefore the age of 65. Cognitive areas affected by dementia include,without limitation, memory, attention span, language, and problemsolving. Generally, symptoms must be present for at least six months tobefore an individual is diagnosed with dementia.

Exemplary forms of dementia include, without limitation, frontotemporaldementia, Alzheimer's disease, vascular dementia, semantic dementia, anddementia with Lewy bodies.

Without wishing to be bound by theory, it is believed that administeringan anti-TREM2 and/or anti-DAP12 antibody of the present disclosure canprevent, reduce the risk, and/or treat dementia. In some embodiments,administering an anti-TREM2 and/or anti-DAP12 antibody may induce one ormore TREM2 and/or DAP12 activities in an individual having dementia(e.g., DAP12 phosphorylation, PI3K activation, increased expression ofone or more anti-inflammatory mediators, or reduced expression of one ormore pro-inflammatory mediators).

Frontotemporal Dementia

Frontotemporal dementia (FTD) is a condition resulting from theprogressive deterioration of the frontal lobe of the brain. Over time,the degeneration may advance to the temporal lobe. Second only toAlzheimer's disease (AD) in prevalence, FTD accounts for 20% ofpre-senile dementia cases. The clinical features of FTD include memorydeficits, behavioral abnormalities, personality changes, and languageimpairments (Cruts, M. & Van Broeckhoven, C., Trends Genet. 24:186-194(2008); Neary, D., et al., Neurology 51:1546-1554 (1998); Ratnavalli,E., Brayne, C., Dawson, K. & Hodges, J. R., Neurology 58:1615-1621(2002)).

A substantial portion of FTD cases are inherited in an autosomaldominant fashion, but even in one family, symptoms can span a spectrumfrom FTD with behavioral disturbances, to Primary Progressive Aphasia,to Cortico-Basal Ganglionic Degeneration. FTD, like mostneurodegenerative diseases, can be characterized by the pathologicalpresence of specific protein aggregates in the diseased brain.Historically, the first descriptions of FTD recognized the presence ofintraneuronal accumulations of hyperphosphorylated Tau protein inneurofibrillary tangles or Pick bodies. A causal role for themicrotubule associated protein Tau was supported by the identificationof mutations in the gene encoding the Tau protein in several families(Hutton, M., et al., Nature 393:702-705 (1998). However, the majority ofFTD brains show no accumulation of hyperphosphorylated Tau but doexhibit immunoreactivity to ubiquitin (Ub) and TAR DNA binding protein(TDP43) (Neumann, M., et al., Arch. Neurol. 64:1388-1394 (2007)). Amajority of those FTD cases with Ub inclusions (FTD-U) were shown tocarry mutations in the progranulin gene.

Without wishing to be bound by theory, it is believed that administeringan anti-TREM2 and/or anti-DAP12 antibody of the present disclosure canprevent, reduce the risk, and/or treat FTD. In some embodiments,administering an anti-TREM2 and/or anti-DAP12 antibody may induce one ormore TREM2 and/or DAP12 activities in an individual having FTD (e.g.,DAP12 phosphorylation, PI3K activation, increased expression of one ormore anti-inflammatory mediators, or reduced expression of one or morepro-inflammatory mediators).

Alzheimer's Disease

Alzheimer's disease (AD) is the most common form of dementia. There isno cure for the disease, which worsens as it progresses, and eventuallyleads to death. Most often, AD is diagnosed in people over 65 years ofage. However, the less-prevalent early-onset Alzheimer's can occur muchearlier.

Common symptoms of Alzheimer's disease include, behavioral symptoms,such as difficulty in remembering recent events; cognitive symptoms,confusion, irritability and aggression, mood swings, trouble withlanguage, and long-term memory loss. As the disease progresses bodilyfunctions are lost, ultimately leading to death. Alzheimer's diseasedevelops for an unknown and variable amount of time before becomingfully apparent, and it can progress undiagnosed for years.

Without wishing to be bound by theory, it is believed that administeringan anti-TREM2 and/or anti-DAP12 antibody of the present disclosure canprevent, reduce the risk, and/or treat Alzheimer's disease. In someembodiments, administering an anti-TREM2 and/or anti-DAP12 antibody mayinduce one or more TREM2 and/or DAP12 activities in an individual havingAlzheimer's disease (e.g., DAP12 phosphorylation, PI3K activation,increased expression of one or more anti-inflammatory mediators, orreduced expression of one or more pro-inflammatory mediators).

Nasu-Hakola Disease

Nasu-Hakola disease (NHD), which may alternatively be referred to aspolycystic lipomembranous osteodysplasia with sclerosingleukoencephalopathy (PLOSL), is a rare inherited leukodystrophycharacterized by progressive presenile dementia associated withrecurrent bone fractures due to polycystic osseous lesions of the lowerand upper extremities. NHD disease course is generally divided into fourstages: latent, osseous, early neurologic, and late neurologic. After anormal development during childhood (latent stage), NHD startsmanifesting during adolescence or young adulthood (typical age of onset20-30 years) with pain in the hands, wrists, ankles, and feet. Patientsthen start suffering from recurrent bone fractures due to polycysticosseous and osteroporotic lesions in the limb bones (osseous stage).During the third or fourth decade of life (early neurologic stage),patients present with pronounced personality changes (e.g., euphoria,lack of concentration, loss of judgment, and social inhibitions)characteristic of a frontal lobe syndrome. Patients also typicallysuffer from progressive memory disturbances. Epileptic seizures are alsofrequently observed. Finally (late neurologic stage), patients progressto a profound dementia, are unable to speak and move, and usually die bythe age of 50.

Without wishing to be bound by theory, it is believed that administeringan anti-TREM2 and/or anti-DAP12 antibody of the present disclosure canprevent, reduce the risk, and/or treat Nasu-Hakola disease (NHD). Insome embodiments, administering an anti-TREM2 and/or anti-DAP12 antibodymay induce one or more TREM2 and/or DAP12 activities in an individualhaving NHD (e.g., DAP12 phosphorylation, PI3K activation, increasedexpression of one or more anti-inflammatory mediators, or reducedexpression of one or more pro-inflammatory mediators).

Parkinson's Disease

Parkinson's disease, which may be referred to as idiopathic or primaryparkinsonism, hypokinetic rigid syndrome (HRS), or paralysis agitans, isa neurodegenerative brain disorder that affects motor system control.The progressive death of dopamine-producing cells in the brain leads tothe major symptoms of Parkinson's. Most often, Parkinson's disease isdiagnosed in people over 50 years of age. Parkinson's disease isidiopathic (having no known cause) in most people. However, geneticfactors also play a role in the disease.

Symptoms of Parkinson's disease include, without limitation, tremors ofthe hands, arms, legs, jaw, and face, muscle rigidity in the limbs andtrunk, slowness of movement (bradykinesia), postural instability,difficulty walking, neuropsychiatric problems, changes in speech orbehavior, depression, anxiety, pain, psychosis, dementia,hallucinations, and sleep problems.

Without wishing to be bound by theory, it is believed that administeringan anti-TREM2 antibody of the present disclosure can prevent, reduce therisk, and/or treat Parkinson's disease. In some embodiments,administering an anti-TREM2 and/or anti-DAP12 antibody may induce one ormore TREM2 and/or DAP12 activities in an individual having Parkinson'sdisease (e.g., DAP12 phosphorylation, PI3K activation, increasedexpression of one or more anti-inflammatory mediators, or reducedexpression of one or more pro-inflammatory mediators).

Amyotrophic Lateral Sclerosis

As used herein, amyotrophic lateral sclerosis (ALS) or, motor neurondisease or, Lou Gehrig's disease are used interchangeably and refer to adebilitating disease with varied etiology characterized by rapidlyprogressive weakness, muscle atrophy and fasciculations, musclespasticity, difficulty speaking (dysarthria), difficulty swallowing(dysphagia), and difficulty breathing (dyspnea).

It has been shown that progranulin play a role in ALS (Schymick, J C etal., (2007) J Neurol Neurosurg Psychiatry.; 78:754-6) and protects againthe damage caused by ALS causing proteins such as TDP-43 (Laird, A S etal., (2010). PLoS ONE 5: e13368). It was also demonstrated that pro-NGFinduces p75 mediated death of oligodendrocytes and corticospinal neuronsfollowing spinal cord injury (Beatty et al., Neuron (2002), 36, pp.375-386; Giehl et al, Proc. Natl. Acad. Sci USA (2004), 101, pp6226-30).

Without wishing to be bound by theory, it is believed that administeringan anti-TREM2 antibody of the present disclosure can prevent, reduce therisk, and/or treat ALS. In some embodiments, administering an anti-TREM2and/or anti-DAP12 antibody may induce one or more TREM2 and/or DAP12activities in an individual having ALS (e.g., DAP12 phosphorylation,PI3K activation, increased expression of one or more anti-inflammatorymediators, or reduced expression of one or more pro-inflammatorymediators).

Huntington's Disease

Huntington's disease (HD) is an inherited neurodegenerative diseasecaused by an autosomal dominant mutation in the Huntingtin gene (HTT).Expansion of a cytokine-adenine-guanine (CAG) triplet repeat within theHuntingtin gene results in production of a mutant form of the Huntingtinprotein (Htt) encoded by the gene. This mutant Huntingtin protein (mHtt)is toxic and contributes to neuronal death. Symptoms of Huntington'sdisease most commonly appear between the ages of 35 and 44, althoughthey can appear at any age.

Symptoms of Huntington's disease, include, without limitation, motorcontrol problems, jerky, random movements (chorea), abnormal eyemovements, impaired balance, seizures, difficulty chewing, difficultyswallowing, cognitive problems, altered speech, memory deficits,thinking difficulties, insomnia, fatigue, dementia, changes inpersonality, depression, anxiety, and compulsive behavior.

Without wishing to be bound by theory, it is believed that administeringan anti-TREM2 antibody of the present disclosure can prevent, reduce therisk, and/or treat Huntington's disease (HD). In some embodiments,administering an anti-TREM2 and/or anti-DAP12 antibody may induce one ormore TREM2 and/or DAP12 activities in an individual having HD (e.g.,DAP12 phosphorylation, PI3K activation, increased expression of one ormore anti-inflammatory mediators, or reduced expression of one or morepro-inflammatory mediators).

Taupathy Disease

Taupathy diseases, or Tauopathies, are a class of neurodegenerativedisease caused by aggregation of the microtubule-associated protein tauwithin the brain. Alzheimer's disease (AD) is the most well-knownTaupathy disease, and involves an accumulation of tau protein withinneurons in the form of insoluble neurofibrillary tangles (NFTs). OtherTaupathy diseases and disorders include progressive supranuclear palsy,dementia pugilistica (chromic traumatic encephalopathy), Frontotemporaldementia and parkinsonism linked to chromosome 17, Lytico-Bodig disease(Parkinson-dementia complex of Guam), Tangle-predominant dementia,Ganglioglioma and gangliocytoma, Meningioangiomatosis, Subacutesclerosing panencephalitis, lead encephalopathy, tuberous sclerosis,Hallervorden-Spatz disease, lipofuscinosis, Pick's disease, corticobasaldegeneration, Argyrophilic grain disease (AGD), Huntington's disease,frontotemporal dementia, and frontotemporal lobar degeneration.

Without wishing to be bound by theory, it is believed that administeringan anti-TREM2 antibody of the present disclosure can prevent, reduce therisk, and/or treat Taupathy disease. In some embodiments, administeringan anti-TREM2 and/or anti-DAP12 antibody may induce one or more TREM2and/or DAP12 activities in an individual having Taupathy disease (e.g.,DAP12 phosphorylation, PI3K activation, increased expression of one ormore anti-inflammatory mediators, or reduced expression of one or morepro-inflammatory mediators).

Multiple Sclerosis

Multiple sclerosis (MS) can also be referred to as disseminatedsclerosis or encephalomyelitis disseminata. MS is an inflammatorydisease in which the fatty myelin sheaths around the axons of the brainand spinal cord are damaged, leading to demyelination and scarring aswell as a broad spectrum of signs and symptoms. MS affects the abilityof nerve cells in the brain and spinal cord to communicate with eachother effectively. Nerve cells communicate by sending electrical signalscalled action potentials down long fibers called axons, which arecontained within an insulating substance called myelin. In MS, thebody's own immune system attacks and damages the myelin. When myelin islost, the axons can no longer effectively conduct signals. MS onsetusually occurs in young adults, and is more common in women.

Symptoms of MS include, without limitation, changes in sensation, suchas loss of sensitivity or tingling; pricking or numbness, such ashypoesthesia and paresthesia; muscle weakness; clonus; muscle spasms;difficulty in moving; difficulties with coordination and balance, suchas ataxia; problems in speech, such as dysarthria, or in swallowing,such as dysphagia; visual problems, such as nystagmus, optic neuritisincluding phosphenes, and diplopia; fatigue; acute or chronic pain; andbladder and bowel difficulties; cognitive impairment of varying degrees;emotional symptoms of depression or unstable mood; Uhthoff's phenomenon,which is an exacerbation of extant symptoms due to an exposure to higherthan usual ambient temperatures; and Lhermitte's sign, which is anelectrical sensation that runs down the back when bending the neck.

Without wishing to be bound by theory, it is believed that administeringan anti-TREM2 and/or anti-DAP12 antibody of the present disclosure canprevent, reduce the risk, and/or treat multiple sclerosis. In someembodiments, administering an anti-TREM2 and/or anti-DAP12 antibody mayinduce one or more TREM2 and/or DAP12 activities in an individual havingmultiple sclerosis (e.g., DAP12 phosphorylation, PI3K activation,increased expression of one or more anti-inflammatory mediators, andreduced expression of one or more pro-inflammatory mediators).

Cancer

Yet further aspects of the present disclosure provide methods forpreventing, reducing risk, or treating an individual having cancer,comprising administering to the individual a therapeutically effectiveamount of an isolated anti-TREM2 and/or anti-DAP12 antibody of thepresent disclosure. Any of the isolated antibodies of the presentdisclosure may be used in these methods. In some embodiments, theisolated antibody is an agonist antibody of the present disclosure. Inother embodiments, the isolated antibody is an antagonist antibody ofthe present disclosure.

As described above, the tumor microenvironment is known to contain aheterogeneous immune infiltrate, which includes T lymphocytes,macrophages and cells of myeloid/granulocytic lineage. In particular,the presence of M2-macrophages in tumors is associated with poorprognosis. Therapies that reduce the number of these cells in the tumor,such as CSF1R blocking agents, are showing beneficial effects inpreclinical models and early stage clinical studies. It has been shownthat TREM2 synergizes with CSF1 to promote survival of macrophages invitro, and that this effect is particularly prominent in M2-typemacrophages, compared to other types of phagocytic cells. A seminalpreclinical study has also shown synergies between drugs that targettumor-associated macrophages (e.g., CSF1/CSF1R blocking antibodies) andcheckpoint blocking antibodies that target T cells, indicating thatmanipulating both cell types shows efficacy in tumor models whereindividual therapies are poorly effective (Zhu Y; Cancer Res. 2014 Sep.15; 74(18):5057-69). Therefore, without wishing to be bound by theory,it is thought that blocking TREM2 signaling in tumor associatedmacrophages may inhibit suppression of the immune response in the tumormicroenvironment, resulting in a therapeutic anti-tumor immune response.

Due to the synergies between TREM2 and CSF1, and between targetingtumor-associated macrophages and targeting T cells, in some embodiments,the methods for preventing, reducing risk, or treating an individualhaving cancer further include administering to the individual at leastone antibody that specifically binds to an inhibitory checkpointmolecule. Examples of antibodies that specifically bind to an inhibitorycheckpoint molecule include, without limitation, an anti-PD-L1 antibody,an anti-CTLA4 antibody, an anti-PD-L2 antibody, an anti-PD-1 antibody,an anti-B7-H3 antibody, an anti-B7-H4 antibody, and anti-HVEM antibody,an anti-BTLA antibody, an anti-GAL9 antibody, an anti-TIM3 antibody, ananti-A2AR antibody, an anti-LAG-3 antibody, an anti-phosphatidylserineantibody, and any combination thereof. In some embodiments, the at leastone antibody that specifically binds to an inhibitory checkpointmolecule is administered in combination with an antagonist anti-TREM2and/or anti-DAP12 antibody of the present disclosure.

In some embodiments, a cancer to be prevented or treated by the methodsof the present disclosure includes, but is not limited to, squamous cellcancer (e.g., epithelial squamous cell cancer), lung cancer includingsmall-cell lung cancer, non-small cell lung cancer, adenocarcinoma ofthe lung and squamous carcinoma of the lung, cancer of the peritoneum,hepatocellular cancer, gastric or stomach cancer includinggastrointestinal cancer and gastrointestinal stromal cancer, pancreaticcancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer,bladder cancer, cancer of the urinary tract, hepatoma, breast cancer,colon cancer, rectal cancer, colorectal cancer, endometrial or uterinecarcinoma, salivary gland carcinoma, kidney or renal cancer, prostatecancer, vulval cancer, thyroid cancer, hepatic carcinoma, analcarcinoma, penile carcinoma, melanoma, superficial spreading melanoma,lentigo maligna melanoma, acral lentiginous melanomas, nodularmelanomas, multiple myeloma and B-cell lymphoma; chronic lymphocyticleukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia;chronic myeloblastic leukemia; and post-transplant lymphoproliferativedisorder (PTLD), as well as abnormal vascular proliferation associatedwith phakomatoses, edema (such as that associated with brain tumors),Meigs' syndrome, brain, as well as head and neck cancer, and associatedmetastases. In some embodiments, the cancer is colorectal cancer. Insome embodiments, the cancer is selected from non-small cell lungcancer, glioblastoma, neuroblastoma, renal cell carcinoma, bladdercancer, ovarian cancer, melanoma, breast carcinoma, gastric cancer, andhepatocellular carcinoma. In some embodiments, the cancer istriple-negative breast carcinoma. In some embodiments, the cancer may bean early stage cancer or a late stage cancer. In some embodiments, thecancer may be a primary tumor. In some embodiments, the cancer may be ametastatic tumor at a second site derived from any of the above types ofcancer.

In some embodiments, anti-TREM2 and/or anti-DAP12 antibodies of thepresent disclosure may be used for preventing, reducing risk, ortreating cancer, including, without limitation, bladder cancer breastcancer, colon and rectal cancer, endometrial cancer, kidney cancer,renal cell cancer, renal pelvis cancer, leukemia, lung cancer, melanoma,non-Hodgkin's lymphoma, pancreatic cancer, prostate cancer, ovariancancer, fibrosarcoma, and thyroid cancer.

In some embodiments, the present disclosure provides methods ofpreventing, reducing risk, or treating an individual having cancer, byadministering to the individual a therapeutically effective amount of ananti-TREM2 and/or anti-DAP12 antibody of the present disclosure.

In some embodiments, the method further includes administering to theindividual at least one antibody that specifically binds to aninhibitory checkpoint molecule, and/or another standard orinvestigational anti-cancer therapy. In some embodiments, the at leastone antibody that specifically binds to an inhibitory checkpointmolecule is administered in combination with the isolated antibody. Insome embodiments, the at least one antibody that specifically binds toan inhibitory checkpoint molecule is selected from an anti-PD-Lantibody, an anti-CTLA4 antibody, an anti-PD-L2 antibody, an anti-PD-1antibody, an anti-B7-H3 antibody, an anti-B7-H4 antibody, and anti-HVEMantibody, an anti-B- and T-lymphocyte attenuator (BTLA) antibody, ananti-Killer inhibitory receptor (KIR) antibody, an anti-GAL9 antibody,an anti-TIM3 antibody, an anti-A2AR antibody, an anti-LAG-3 antibody, ananti-phosphatidylserine antibody, an anti-CD27 antibody, and anycombination thereof. In some embodiments, the standard orinvestigational anti-cancer therapy is one or more therapies selectedfrom radiotherapy, cytotoxic chemotherapy, targeted therapy, imatinib(Gleevec®), trastuzumab (Herceptin®), adoptive cell transfer (ACT),chimeric antigen receptor T cell transfer (CAR-T), vaccine therapy, andcytokine therapy.

In some embodiments, the method further includes administering to theindividual at least one antibody that specifically binds to aninhibitory cytokine. In some embodiments, the at least one antibody thatspecifically binds to an inhibitory cytokine is administered incombination with the isolated antibody. In some embodiments, the atleast one antibody that specifically binds to an inhibitory cytokine isselected from an anti-CCL2 antibody, an anti-CSF-1 antibody, ananti-IL-2 antibody, and any combination thereof.

In some embodiments, the method further includes administering to theindividual at least one agonistic antibody that specifically binds to astimulatory checkpoint protein. In some embodiments, the at least oneagonistic antibody that specifically binds to a stimulatory checkpointprotein is administered in combination with the isolated antibody. Insome embodiments, the at least one agonistic antibody that specificallybinds to a stimulatory checkpoint protein is selected from an agonistanti-CD40 antibody, an agonist anti-OX40 antibody, an agonist anti-ICOSantibody, an agonist anti-CD28 antibody, an agonist anti-CD137/4-1BBantibody, an agonist anti-CD27 antibody, an agonistanti-glucocorticoid-induced TNFR-related protein GITR antibody, and anycombination thereof.

In some embodiments, the method further includes administering to theindividual at least one stimulatory cytokine. In some embodiments, theat least one stimulatory cytokine is administered in combination withthe isolated antibody. In some embodiments, the at least one stimulatorycytokine is selected from TNF-α, IL-10, IL-6, IL-8, CRP, TGF-betamembers of the chemokine protein families, IL20 family member, IL-33,LIF, OSM, CNTF, TGF-beta, IL-11, IL-12, IL-17, IL-8, CRP, IFN-α, IFN-β,IL-2, IL-18, GM-CSF, G-CSF, and any combination thereof.

Kits/Articles of Manufacture

The present disclosure also provides kits containing an isolatedantibody of the present disclosure (e.g., an anti-TREM2 or anti-DAP12antibody described herein), or a functional fragment thereof. Kits ofthe present disclosure may include one or more containers comprising apurified antibody of the present disclosure. In some embodiments, thekits further include instructions for use in accordance with the methodsof this disclosure. In some embodiments, these instructions comprise adescription of administration of the isolated antibody of the presentdisclosure (e.g., an anti-TREM2 or anti-DAP12 antibody described herein)to prevent, reduce risk, or treat an individual having a disease,disorder, or injury selected from dementia, frontotemporal dementia,Alzheimer's disease, Nasu-Hakola disease, and multiple sclerosis,according to any methods of this disclosure.

In some embodiments, the instructions comprise a description of how todetect TREM2 and/or DAP12, for example in an individual, in a tissuesample, or in a cell. The kit may further comprise a description ofselecting an individual suitable for treatment based on identifyingwhether that individual has the disease and the stage of the disease.

In some embodiments, the kits may further include another antibody ofthe present disclosure (e.g., at least one antibody that specificallybinds to an inhibitory checkpoint molecule, at least one antibody thatspecifically binds to an inhibitory cytokine, and/or at least oneagonistic antibody that specifically binds to a stimulatory check pointprotein) and/or at least one stimulatory cytokine. In some embodiments,the kits may further include instructions for using the antibody and/orstimulatory cytokine in combination with an isolated antibody of thepresent disclosure (e.g., an anti-TREM2 antagonist antibody describedherein), instructions for using the isolated antibody of the presentdisclosure in combination with an antibody and/or stimulatory cytokine,or instructions for using an isolated antibody of the present disclosureand an antibody and/or stimulatory cytokine, according to any methods ofthis disclosure.

The instructions generally include information as to dosage, dosingschedule, and route of administration for the intended treatment. Thecontainers may be unit doses, bulk packages (e.g., multi-dose packages)or sub-unit doses. Instructions supplied in the kits of the presentdisclosure are typically written instructions on a label or packageinsert (e.g., a paper sheet included in the kit), but machine-readableinstructions (e.g., instructions carried on a magnetic or opticalstorage disk) are also acceptable.

The label or package insert indicates that the composition is used fortreating, e.g., a disease of the present disclosure. Instructions may beprovided for practicing any of the methods described herein.

The kits of this disclosure are in suitable packaging. Suitablepackaging includes, but is not limited to, vials, bottles, jars,flexible packaging (e.g., sealed Mylar or plastic bags), and the like.Also contemplated are packages for use in combination with a specificdevice, such as an inhaler, nasal administration device (e.g., anatomizer) or an infusion device such as a minipump. A kit may have asterile access port (for example the container may be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). The container may also have a sterile access port(e.g., the container may be an intravenous solution bag or a vial havinga stopper pierceable by a hypodermic injection needle). At least oneactive agent in the composition is an isolated antibody of the presentdisclosure (e.g., an anti-TREM2 or anti-DAP12 antibody describedherein). The container may further comprise a second pharmaceuticallyactive agent.

Kits may optionally provide additional components such as buffers andinterpretive information. Normally, the kit comprises a container and alabel or package insert(s) on or associated with the container.

Diagnostic Uses

The isolated antibodies of the present disclosure (e.g., an anti-TREM2or anti-DAP12 antibody described herein) also have diagnostic utility.This disclosure therefore provides for methods of using the antibodiesof this disclosure, or functional fragments thereof, for diagnosticpurposes, such as the detection of TREM2 and/or DAP12 in an individualor in tissue samples derived from an individual.

In some embodiments, the individual is a human. In some embodiments, theindividual is a human patient suffering from, or at risk for developingdementia, frontotemporal dementia, Alzheimer's disease, Nasu-Hakoladisease, Parkinson's disease, Amyotrophic lateral sclerosis,Huntington's disease, Taupathy disease, multiple sclerosis, or cancer.In some embodiments, the diagnostic methods involve detecting TREM2and/or DAP12 in a biological sample, such as a biopsy specimen, atissue, or a cell. An isolated antibody of the present disclosure (e.g.,an anti-TREM2 or anti-DAP12 antibody described herein) is contacted withthe biological sample and antigen-bound antibody is detected. Forexample, a tissue sample (e.g., a biopsy specimen) may be stained withan anti-TREM2 or anti-DAP12 antibody described herein in order to detectand/or quantify disease-associated macrophages (e.g., M2-typemacrophages). The detection method may involve quantification of theantigen-bound antibody. Antibody detection in biological samples mayoccur with any method known in the art, including immunofluorescencemicroscopy, immunocytochemistry, immunohistochemistry, ELISA, FACSanalysis, immunoprecipitation, or micro-positron emission tomography. Incertain embodiments, the antibody is radiolabeled, for example with 8Fand subsequently detected utilizing micro-positron emission tomographyanalysis. Antibody-binding may also be quantified in a patient bynon-invasive techniques such as positron emission tomography (PET),X-ray computed tomography, single-photon emission computed tomography(SPECT), computed tomography (CT), and computed axial tomography (CAT).

In other embodiments, an isolated antibody of the present disclosure(e.g., an anti-TREM2 or anti-DAP12 antibody described herein) may beused to detect and/or quantify, for example, microglia in a brainspecimen taken from a preclinical disease model (e.g., a non-humandisease model). As such, an isolated antibody of the present disclosure(e.g., an anti-TREM2 or anti-DAP12 antibody described herein) may beuseful in evaluating therapeutic response after treatment in a model fora nervous system disease or injury such as dementia, frontotemporaldementia, Alzheimer's disease, Nasu-Hakola disease, or multiplesclerosis, as compared to a control.

ENUMERATED EMBODIMENTS

The following enumerated embodiments are representative of some aspectsof the invention.

1. An isolated agonist antibody that binds to a TREM2 protein, a DAP12protein, or both, wherein the antibody induces one or more TREM2activities, DAP12 activities, or both.2. The isolated antibody of embodiment 1, wherein the TREM2 protein, theDAP12 protein, or both is a mammalian protein or a human protein.3. The isolated antibody of embodiment 2, wherein the TREM2 protein, theDAP12 protein, or both is a wild-type protein.4. The isolated antibody of embodiment 2, wherein the TREM2 protein, theDAP12 protein, or both is a naturally occurring variant.5. The isolated antibody of any one of embodiments 1-4, wherein the oneor more TREM2 activities comprise TREM2 binding to DAP12.6. The isolated antibody of any one of embodiments 1-4, wherein the oneor more DAP12 activities comprise DAP12 binding to TREM2.7. The isolated antibody of any one of embodiments 1-6, wherein the oneor more TREM2 activities, DAP12 activities, or both comprise DAP12phosphorylation.8. The isolated antibody of embodiment 7, wherein DAP12 phosphorylationis induced by one or more SRC family tyrosine kinases.9. The isolated antibody of any one of embodiments 1-8, wherein the oneor more TREM2 activities, DAP12 activities, or both comprise PI3Kactivation.10. The isolated antibody of any one of embodiments 1-9, wherein the oneor more TREM2 activities, DAP12 activities, or both comprise increasedexpression of one or more anti-inflammatory mediators selected from thegroup consisting of IL-12p70, IL-6, and IL-10.11. The isolated antibody of embodiment 10, wherein the increasedexpression of the one or more anti-inflammatory mediators occurs in oneor more cells selected from the group consisting of macrophages,dendritic cells, and microglial cells.12. The isolated antibody of any one of embodiments 1-11, wherein theone or more TREM2 activities, DAP12 activities, or both comprise reducedexpression of one or more pro-inflammatory mediators selected from thegroup consisting of IFN-a4, IFN-b, IL-6, IL-12 p70, IL-1β and TNF.13. The isolated antibody of embodiment 12, wherein the reducedexpression of the one or more pro-inflammatory mediators occurs in oneor more cells selected from the group consisting of macrophages,dendritic cells, and microglial cells.14. The isolated antibody of any one of embodiments 1-13, wherein theone or more TREM2 activities, DAP12 activities, or both compriseextracellular signal-regulated kinase (ERK) phosphorylation.15. The isolated antibody of any one of embodiments 1-14, wherein theone or more TREM2 activities, DAP12 activities, or both compriseincreased expression of C-C chemokine receptor 7 (CCR7).16. The isolated antibody of any one of embodiments 1-15, wherein theone or more TREM2 activities, DAP12 activities, or both compriseinduction of microglial cell chemotaxis toward CCL19 and CCL21expressing cells.17. The isolated antibody of any one of embodiments 1-16, wherein theone or more TREM2 activities, DAP12 activities, or both comprise areduction, normalization, or both of the ability of bone marrow-deriveddendritic cells to induce antigen-specific T-cell proliferation.18. The isolated antibody of any one of embodiments 1-17, wherein theone or more TREM2 activities, DAP12 activities, or both compriseinduction of osteoclast production, increased rate ofosteoclastogenesis, or both.19. The isolated antibody of any one of embodiments 1-18, wherein theone or more TREM2 activities, DAP12 activities, or both comprise ofincreasing the survival of macrophages, microglial cells, or both.20. The isolated antibody of any one of embodiments 1-19, wherein theone or more TREM2 activities, DAP12 activities, or both compriseincreasing the function of macrophages, microglial cells, or both.21. The isolated antibody of any one of any one of embodiments 1-20,wherein the one or more TREM2 activities, DAP12 activities, or bothcomprise induction of one or more types of clearance withoutinflammation selected from the group consisting of apoptotic neuronclearance without inflammation, nerve tissue debris clearance withoutinflammation, non-nerve tissue debris clearance without inflammation,bacteria or other foreign body clearance without inflammation, anddisease-causing protein clearance without inflammation.22. The isolated antibody of any one of embodiments 1-21, wherein theone or more TREM2 activities, DAP12 activities, or both compriseinduction of phagocytosis without inflammation of one or more ofapoptotic neurons, nerve tissue debris, non-nerve tissue debris,bacteria, other foreign bodies, or disease-causing proteins withoutinflammation.23. The isolated antibody of embodiment 21 or embodiment 22, wherein thedisease-causing protein is selected from the group consisting of A betapeptide, alpha synuclain protein, Tau protein, TDP-43 protein, prionprotein, and huntingtin protein.24. The isolated antibody of any one of embodiments 1-23, wherein theone or more TREM2 activities, DAP12 activities, or both comprisenormalization of disrupted TREM2/DAP12-dependent gene expression.25. The isolated antibody of any one of embodiments 1-24, wherein theone or more TREM2 activities, DAP12 activities, or both compriserecruitment of Syk, ZAP70, or both to DAP12.26. The isolated antibody of any one of embodiments 1-25, wherein theisolated agonist antibody that binds to a TREM2 protein binds to one ormore amino acids within amino acid residues selected from the groupconsisting of:

-   -   i. amino acid residues 29-112 of SEQ ID NO: 1, or amino acid        residues on a TREM2 protein corresponding to amino acid residues        29-112 of SEQ ID NO: 1;    -   ii. amino acid residues 29-41 of SEQ ID NO: 1, or amino acid        residues on a TREM2 protein corresponding to amino acid residues        29-41 of SEQ ID NO: 1;    -   iii. amino acid residues 40-44 of SEQ ID NO: 1, or amino acid        residues on a TREM2 protein corresponding to amino acid residues        40-44 of SEQ ID NO: 1;    -   iv. amino acid residues 47-69 of SEQ ID NO: 1, or amino acid        residues on a TREM2 protein corresponding to amino acid residues        47-69 of SEQ ID NO: 1;    -   v. amino acid residues 67-76 of SEQ ID NO: 1, or amino acid        residues on a TREM2 protein corresponding to amino acid residues        67-76 of SEQ ID NO: 1;    -   vi. amino acid residues 76-86 of SEQ ID NO: 1, or amino acid        residues on a TREM2 protein corresponding to amino acid residues        76-86 of SEQ ID NO: 1;    -   vii. amino acid residues 91-100 of SEQ ID NO: 1, or amino acid        residues on a TREM2 protein corresponding to amino acid residues        91-100 of SEQ ID NO: 1;    -   viii. amino acid residues 99-115 of SEQ ID NO: 1, or amino acid        residues on a TREM2 protein corresponding to amino acid residues        99-115 of SEQ ID NO: 1;    -   ix. amino acid residues 104-112 of SEQ ID NO: 1, or amino acid        residues on a TREM2 protein corresponding to amino acid residues        104-112 of SEQ ID NO: 1; and    -   x. amino acid residues 114-118 of SEQ ID NO: 1, or amino acid        residues on a TREM2 protein corresponding to amino acid residues        114-118 of SEQ ID NO: 1.        27. The isolated antibody of any one of embodiments 1-25,        wherein the isolated agonist antibody that binds to a TREM2        protein binds to an epitope comprising one or more amino acid        residues selected from the group consisting of:    -   i. amino acid residue Arg47 or Asp87 of SEQ ID NO: 1;    -   ii. amino acid residues 40-44 of SEQ ID NO: 1;    -   iii. amino acid residues 67-76 of SEQ ID NO: 1; and    -   iv. amino acid residues 114-118 of SEQ ID NO: 1.        28. The isolated antibody of any one of embodiments 1-25,        wherein the isolated agonist antibody that binds to a DAP12        protein binds to one or more amino acids within amino acid        residues 22-40 of SEQ ID NO: 2, or amino acid residues on a        DAP12 protein corresponding to amino acid residues 22-40 of SEQ        ID NO: 2.        29. The isolated antibody of any one of embodiments 1-25,        wherein the isolated agonist antibody is a bispecific antibody        that binds to one or more amino acids selected from the group        consisting of:    -   i. one or more amino acid residues of SEQ ID NO: 1, or amino        acid residues on a TREM2 protein corresponding to amino acid        residues of SEQ ID NO: 1; and    -   ii. one or more amino acid residues of SEQ ID NO: 2, or amino        acid residues on a DAP12 protein corresponding to amino acid        residues of SEQ ID NO: 2.        30. The isolated antibody of any of the preceding embodiments,        wherein the antibody is a human antibody, a humanized antibody,        a bispecific antibody, a multivalent antibody, or a chimeric        antibody.        31. The isolated antibody of any of the preceding embodiments,        wherein the antibody is a monoclonal antibody.        32. The isolated antibody of any of the preceding embodiments,        wherein the isolated antibody is an antibody fragment that binds        to one or more human proteins selected from the group consisting        of human TREM2, a naturally occurring variant of human TREM2,        human DAP12, and naturally occurring variant of human DAP12.        33. The isolated antibody of embodiment 32, wherein the fragment        is an Fab, Fab′, Fab′-SH, F(ab′)2, Fv or scFv fragment.        34. An isolated nucleic acid encoding the antibody of any one of        the preceding embodiments.        35. A vector comprising the nucleic acid of embodiment 34.        36. A host cell comprising the vector of embodiment 35.        37. A method of producing an antibody, comprising culturing the        host cells of embodiment 36 so that the antibody is produced.        38. The method of embodiment 37, further comprising recovering        the antibody produced by the host cell.        39. A pharmaceutical composition comprising the antibody of any        one of embodiments 1-33, and a pharmaceutically acceptable        carrier.        40. A method of preventing, reducing risk, or treating an        individual having a disease, disorder, or injury selected from        the group consisting of dementia, frontotemporal dementia,        Alzheimer's disease, Nasu-Hakola disease, and multiple        sclerosis, comprising administering to the individual a        therapeutically effective amount of the isolated antibody of any        one of embodiments 1-33.        41. The method of embodiment 40, wherein the individual has a        heterozygous variant of TREM2, wherein the variant comprises one        or more substitutions selected from the group consisting of:    -   i. a glutamic acid to stop codon substitution in the nucleic        acid sequence encoding amino acid residue Glu14 of SEQ ID NO: 1;    -   ii. a glutamine to stop codon substitution in the nucleic acid        sequence encoding amino acid residue Gln33 of SEQ ID NO: 1;    -   iii. a tryptophan to stop codon substitution in the nucleic acid        sequence encoding amino acid residue Trp44 of SEQ ID NO: 1;    -   iv. an arginine to histidine amino acid substitution at an amino        acid corresponding to amino acid residue Arg47 of SEQ ID NO: 1    -   v. a tryptophan to stop codon substitution in the nucleic acid        sequence encoding amino acid residue Trp78 of SEQ ID NO: 1;    -   vi. a valine to glycine amino acid substitution at an amino acid        corresponding to amino acid residue Val126 of SEQ ID NO: 1;    -   vii. an aspartic acid to glycine amino acid substitution at an        amino acid corresponding to amino acid residue Asp134 of SEQ ID        NO: 1; and    -   viii. a lysine to asparagine amino acid substitution at an amino        acid corresponding to amino acid residue Lys186 of SEQ ID NO: 1.        42. The method of embodiment 40, wherein the individual has a        heterozygous variant of TREM2, wherein the variant comprises a        guanine nucleotide deletion at a nucleotide corresponding to        nucleotide residue G313 of the nucleic acid sequence encoding        SEQ ID NO: 1; a guanine nucleotide deletion at a nucleotide        corresponding to nucleotide residue G267 of the nucleic acid        sequence encoding SEQ ID NO: 1; or both.        43. The method of embodiment 40, wherein the individual has a        heterozygous variant of DAP12, wherein the variant comprises one        or more variants selected from the group consisting of:    -   i. a methionine to threonine substitution at an amino acid        corresponding to amino acid residue Met1 of SEQ ID NO: 2;    -   ii. a glycine to argenine amino acid substitution at an amino        acid corresponding to amino acid residue Gly49 of SEQ ID NO: 2;    -   iii. a deletion within exons 1-4 of the nucleic acid sequence        encoding SEQ ID NO: 2;    -   iv. an insertion of 14 amino acid residues at exon 3 of the        nucleic acid sequence encoding SEQ ID NO: 2; and    -   v. a guanine nucleotide deletion at a nucleotide corresponding        to nucleotide residue G141 of the nucleic acid sequence encoding        SEQ ID NO: 2.        44. An isolated agonist antibody that binds to a TREM2 protein,        a DAP12 protein, or both, wherein the antibody induces one or        more TREM2 activities, DAP12 activities, or both.        45. The isolated antibody of embodiment 44, wherein the TREM2        protein, the DAP12 protein, or both is a mammalian protein or a        human protein.        46. The isolated antibody of embodiment 45, wherein the TREM2        protein, the DAP12 protein, or both is a wild-type protein.        47. The isolated antibody of embodiment 45, wherein the TREM2        protein, the DAP12 protein, or both is a naturally occurring        variant.        48. The isolated antibody of any one of embodiments 44-47,        wherein the isolated antibody induces or retains TREM2        clustering, DAP12 clustering, or both on a cell surface.        49. The isolated antibody of any one of embodiments 44-48,        wherein the one or more TREM2 activities comprise TREM2 binding        to DAP12.        50. The isolated antibody of any one of embodiments 44-48,        wherein the one or more DAP12 activities comprise DAP12 binding        to TREM2.        51. The isolated antibody of any one of embodiments 44-50,        wherein the one or more TREM2 activities, DAP12 activities, or        both comprise DAP12 phosphorylation, TREM2 phosphorylation, or        both.        52. The isolated antibody of embodiment 51, wherein DAP12        phosphorylation, TREM2 phosphorylation, or both is induced by        one or more SRC family tyrosine kinases.        53. The isolated antibody of embodiment 52, wherein the one or        more SRC family tyrosine kinases comprise a Syk kinase.        54. The isolated antibody of any one of embodiments 44-53,        wherein the one or more TREM2 activities, DAP12 activities, or        both comprise PI3K activation.        55. The isolated antibody of any one of embodiments 44-54,        wherein the one or more TREM2 activities, DAP12 activities, or        both comprise increased expression of one or more        anti-inflammatory cytokines.        56. The isolated antibody of any one of embodiments 44-54,        wherein the one or more TREM2 activities, DAP12 activities, or        both comprise increased expression of one or more        anti-inflammatory mediators selected from the group consisting        of IL-12p70, IL-6, and IL-10.        57. The isolated antibody of embodiment 55 or embodiment 56,        wherein the increased expression occurs in one or more cells        selected from the group consisting of macrophages, dendritic        cells, monocytes, osteoclasts, Langerhans cells of skin, Kupffer        cells, and microglial cells.        58. The isolated antibody of any one of embodiments 44-57,        wherein the one or more TREM2 activities, DAP12 activities, or        both comprise reduced expression of one or more pro-inflammatory        cytokines.        59. The isolated antibody of any one of embodiments 44-57,        wherein the one or more TREM2 activities, DAP12 activities, or        both comprise reduced expression of one or more pro-inflammatory        mediators selected from the group consisting of IFN-a4, IFN-b,        IL-6, IL-12 p70, IL-1β TNF, TNF-α, IL-10, IL-8, CRP, TGF-beta        members of the chemokine protein families, IL-20 family members,        IL-33, LIF, IFN-gamma, OSM, CNTF, TGF-beta, GM-CSF, IL-11,        IL-12, IL-17, IL-18, and CRP.        60. The isolated antibody of any one of embodiments 44-58,        wherein the one or more TREM2 activities, DAP12 activities, or        both comprise reduced expression of TNF-α, IL-6, or both.        61. The isolated antibody of embodiment 58 or embodiment 60,        wherein the reduced expression of the one or more        pro-inflammatory mediators occurs in one or more cells selected        from the group consisting of macrophages, dendritic cells,        dendritic cells, monocytes, osteoclasts, Langerhans cells of        skin, Kupffer cells, and microglial cells.        62. The isolated antibody of any one of embodiments 44-60,        wherein the one or more TREM2 activities, DAP12 activities, or        both comprise extracellular signal-regulated kinase (ERK)        phosphorylation.        63. The isolated antibody of any one of embodiments 44-62,        wherein the one or more TREM2 activities, DAP12 activities, or        both comprise increased expression of C-C chemokine receptor 7        (CCR7).        64. The isolated antibody of any one of embodiments 44-63,        wherein the one or more TREM2 activities, DAP12 activities, or        both comprise induction of microglial cell chemotaxis toward        CCL19 and CCL21 expressing cells.        65. The isolated antibody of any one of embodiments 44-64,        wherein the one or more TREM2 activities, DAP12 activities, or        both comprise an enhancement, normalization, or both of the        ability of bone marrow-derived dendritic cells to induce        antigen-specific T-cell proliferation.        66. The isolated antibody of any one of embodiments 44-65,        wherein the one or more TREM2 activities, DAP12 activities, or        both comprise induction of osteoclast production, increased rate        of osteoclastogenesis, or both.        67. The isolated antibody of any one of embodiments 44-66,        wherein the one or more TREM2 activities, DAP12 activities, or        both comprise increasing the survival of macrophages, microglial        cells, dendritic cells, macrophages, monocytes, osteoclasts,        Langerhans cells of skin, and/or Kupffer cells.        68. The isolated antibody of any one of embodiments 44-67,        wherein the one or more TREM2 activities, DAP12 activities, or        both comprise increasing the function of macrophages, microglial        cells, dendritic cells, macrophages, monocytes, osteoclasts,        Langerhans cells of skin, and/or Kupffer cells.        69. The isolated antibody of embodiment 67 or embodiment 68,        wherein the macrophages and/or microglial cells are M1        macrophages and/or microglial cells, M2 macrophages and/or        microglial cells, or both.        70. The isolated antibody of embodiment 69, wherein the M1        macrophages and/or microglial cells are activated M1 macrophages        and/or microglial cells.        71. The isolated antibody of any one of any one of embodiments        44-68, wherein the one or more TREM2 activities, DAP12        activities, or both comprise induction of one or more types of        clearance selected from the group consisting of apoptotic neuron        clearance, nerve tissue debris clearance, non-nerve tissue        debris clearance, bacteria or other foreign body clearance,        disease-causing protein clearance, disease-causing peptide        clearance, and disease-causing nucleic acid clearance.        72. The isolated antibody of any one of embodiments 44-71,        wherein the one or more TREM2 activities, DAP12 activities, or        both comprise induction of phagocytosis of one or more of        apoptotic neurons, nerve tissue debris, non-nerve tissue debris,        bacteria, other foreign bodies, disease-causing proteins,        disease-causing peptides, or disease-causing nucleic acids.        73. The isolated antibody of embodiment 71 or embodiment 72,        wherein the disease-causing protein is selected from the group        consisting of amyloid beta, Tau, IAPP, alpha-synuclein, TDP-43,        prion protein, PrPSc, huntingtin, calcitonin, superoxide        dismutase, ataxin, Lewy body, atrial natriuretic factor, islet        amyloid polypeptide, insulin, apolipoprotein AI, serum amyloid        A, medin, prolactin, transthyretin, lysozyme, beta 2        microglobulin, gelsolin, keratoepithelin, cystatin,        immunoglobulin light chain AL, S-IBM protein, Repeat-associated        non-ATG (RAN) translation products, DiPeptide repeat (DPR)        peptides, glycine-alanine (GA) repeat peptides, glycine-proline        (GP) repeat peptides, glycine-arginine (GR) repeat peptides,        proline-alanine (PA) repeat peptides, and proline-arginine (PR)        repeat peptides.        74. The isolated antibody of embodiment 71 or embodiment 72,        wherein the disease-causing nucleic acid is antisense GGCCCC        (G2C4) repeat-expansion RNA.        75. The isolated antibody of any one of embodiments 44-74,        wherein the one or more TREM2 activities, DAP12 activities, or        both comprise normalization of disrupted TREM2/DAP12-dependent        gene expression.        76. The isolated antibody of any one of embodiments 44-75,        wherein the one or more TREM2 activities, DAP12 activities, or        both comprise recruitment of Syk, ZAP70, or both to a        DAP12/TREM2 complex.        77. The isolated antibody of any one of embodiments 44-76,        wherein the one or more TREM2 activities, DAP12 activities, or        both comprise Syk phosphorylation.        78. The isolated antibody of any one of embodiments 44-77,        wherein the one or more TREM2 activities, DAP12 activities, or        both comprise increased expression of CD83 and/or CD86 on        dendritic cells.        79. The isolated antibody of any one of embodiments 44-78,        wherein the one or more TREM2 activities, DAP12 activities, or        both comprise reduced secretion of one or more inflammatory        cytokines.        80. The isolated antibody of embodiment 79, wherein the one or        more inflammatory cytokines are selected from the group        consisting of TNF-α, IL-10, IL-6, MCP-1, IFN-a4, IFN-b, IL-1β,        IL-8, CRP, TGF-beta members of the chemokine protein families,        IL-20 family members, IL-33, LIF, IFN-gamma, OSM, CNTF,        TGF-beta, GM-CSF, IL-11, IL-12, IL-17, and IL-18.        81. The isolated antibody of any one of embodiments 44-79,        wherein the one or more TREM2 activities, DAP12 activities, or        both comprise reduced expression of one or more inflammatory        receptors.        82. The isolated antibody of embodiment 81, wherein the one or        more inflammatory receptors comprise CD86.        83. The isolated antibody of any one of embodiments 44-82,        wherein the one or more TREM2 activities, DAP12 activities, or        both comprise increasing phagocytosis by macrophages, dendritic        cells, monocytes, and/or microglia under conditions of reduced        levels of MCSF.        84. The isolated antibody of any one of embodiments 44-82,        wherein the one or more TREM2 activities, DAP12 activities, or        both comprise decreasing phagocytosis by macrophages, dendritic        cells, monocytes, and/or microglia in the presence of normal        levels of MCSF.        85. The isolated antibody of any one of embodiments 44-84,        wherein the one or more TREM2 activities, DAP12 activities, or        both comprise increasing activity of one or more TREM2-dependent        genes.        86. The isolated antibody of embodiment 85, wherein the one or        more TREM2-dependent genes comprise one or more nuclear factor        of activated T-cells (NFAT) transcription factors.        87. The isolated antibody of any one of embodiments 44-86,        wherein the antibody is of the IgG class the IgM class, or the        IgA class.        88. The isolated antibody of embodiment 87, wherein the antibody        is of the IgG class and has an IgG1, IgG2, IgG3, or IgG4        isotype.        89. The isolated antibody of embodiment 87, wherein the antibody        has an IgG2 isotype.        90. The isolated antibody of embodiment 89, wherein the antibody        comprises a human IgG2 constant region.        91. The isolated antibody of embodiment 90, wherein the human        IgG2 constant region comprises an Fc region.        92. The isolated antibody of any one of embodiments 89-91,        wherein the antibody induces the one or more TREM2 activities,        DAP12 activities, or both independently of binding to an Fe        receptor.        93. The isolated antibody of any one of embodiments 89-91,        wherein the antibody binds an inhibitory Fc receptor.        94. The isolated antibody of embodiment 93, wherein the        inhibitory Fc receptor is inhibitory Fc-gamma receptor IIB        (FcγIIB).        95. The isolated antibody of embodiment 93 or embodiment 94,        wherein the human IgG2 constant region comprises an Fc region        that comprises one or more modifications.        96. The isolated antibody of embodiment 95, wherein the Fc        region comprises one or more amino acid substitutions.        97. The isolated antibody of embodiment 96, wherein the one or        more amino acid substitutions in the Fc region are at a residue        position selected from the group consisting of V234A, G237A,        H268Q, V309L, A330S, P331S, C232S, C233S, S267E, L328F, M252Y,        S254T, T256E, and any combination thereof, wherein the numbering        of the residues is according to EU numbering.        98. The isolated antibody of embodiment 90, wherein the human        IgG2 constant region comprises a light chain constant region        comprising a C214S amino acid substitution, wherein the        numbering of the residues is according to EU numbering.        99. The isolated antibody of embodiment 87, wherein the antibody        has an IgG1 isotype.        100. The isolated antibody of embodiment 99, wherein the        antibody comprises a human IgG1 constant region.        101. The isolated antibody of embodiment 100, wherein the human        IgG1 constant region comprises an Fc region.        102. The isolated antibody of any one of embodiments 99-101,        wherein the antibody binds an inhibitory Fc receptor.        103. The isolated antibody of embodiment 102, wherein the        inhibitory Fc receptor is inhibitory Fc-gamma receptor IIB        (FcγRIIB).        104. The isolated antibody of any one of embodiments 101-103,        wherein the Fc region comprises one or more modifications.        105. The isolated antibody of embodiment 104, wherein the Fc        region comprises one or more amino acid substitutions.        106. The isolated antibody of embodiment 105, wherein the one or        more amino acid substitutions in the Fc region are at a residue        position selected from the group consisting of N297A, D265A,        L234A, L235A, G237A, C226S, C229S, E233P, L234V, L234F, L235E,        P331S, S267E, L328F, A330L, M252Y, S254T, T256E, and any        combination thereof, wherein the numbering of the residues is        according to EU numbering.        107. The isolated antibody of any one of embodiments 101-103,        wherein the antibody comprises an IgG2 isotype heavy chain        constant domain 1(CH1) and hinge region.        108. The isolated antibody of embodiment 107, wherein the IgG2        isotype CH1 and hinge region comprise the amino acid sequence of        ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYHFEPVTVS WNSGALTSGVHTFPAVLQSS        GLYSLSSVVT VPSSNFGTQT YTCNVDHKPS NTKVDKTVERKCCVECPPCP (SEQ ID        NO: 397).        109. The isolated antibody of embodiment 107 or embodiment 108,        wherein the antibody Fc region comprises a S267E amino acid        substitution, a L328F amino acid substitution, or both, and/or a        N297A or N297Q amino acid substitution, wherein the numbering of        the residues is according to EU numbering.        110. The isolated antibody of embodiment 99, wherein the        antibody comprises a mouse IgG1 constant region.        111. The isolated antibody of embodiment 87, wherein the        antibody has an IgG4 isotype.        112. The isolated antibody of embodiment 111, wherein the        antibody comprises a human IgG4 constant region.        113. The isolated antibody of embodiment 112, wherein the human        IgG4 constant region comprises an Fc region.        114. The isolated antibody of any one of embodiments 111-113,        wherein the antibody binds an inhibitory Fc receptor.        115. The isolated antibody of embodiment 114, wherein the        inhibitory Fc receptor is inhibitory Fc-gamma receptor IIB        (FcγIIB).        116. The isolated antibody of any one of embodiments 113-115,        wherein the Fc region comprises one or more modifications.        117. The isolated antibody of embodiment 116, wherein the Fc        region comprises one or more amino acid substitutions.        118. The isolated antibody of embodiment 117, wherein the one or        more amino acid substitutions in the Fc region are at a residue        position selected from the group consisting of L235A, G237A,        S228P, L236E, S267E, E318A, L328F, M252Y, S254T, T256E, and any        combination thereof, wherein the numbering of the residues is        according to EU numbering.        119. The isolated antibody of embodiment 87, wherein the        antibody has a hybrid IgG2/4 isotype.        120. The isolated antibody of embodiment 119, wherein the        antibody comprises an amino acid sequence comprising amino acids        118 to 260 of human IgG2 and amino acids 261 to 447 of human        IgG4, wherein the numbering of the residues is according to EU        numbering.        121. The isolated antibody of embodiment 111, wherein the        antibody comprises a mouse IgG4 constant region.        122. The isolated antibody of any one of embodiments 1-121,        wherein the isolated antibody is an antibody fragment that binds        to one or more human proteins selected from the group consisting        of human TREM2, a naturally occurring variant of human TREM2,        human DAP12, and naturally occurring variant of human DAP12, and        wherein the antibody fragment is cross-linked to a second        antibody fragment that binds to one or more human proteins        selected from the group consisting of human TREM2, a naturally        occurring variant of human TREM2, human DAP12, and naturally        occurring variant of human DAP12.        123. The isolated antibody of embodiment 122, wherein the        fragment is an Fab, Fab′, Fab′-SH, F(ab′)2, Fv or scFv fragment.        124. An isolated inert antibody that binds to a TREM2 protein, a        DAP12 protein, or both.        125. An isolated antagonist antibody that binds to a TREM2        protein, a DAP12 protein, or both.        126. The isolated antibody of embodiment 124 or embodiment 125,        wherein the TREM2 protein, the DAP12 protein, or both is a        mammalian protein or a human protein.        127. The isolated antibody of embodiment 126, wherein the TREM2        protein, the DAP12 protein, or both is a wild-type protein.        128. The isolated antibody of embodiment 126, wherein the TREM2        protein, the DAP12 protein, or both is a naturally occurring        variant.        129. The isolated antibody of any one of embodiments 125-128,        wherein the isolated antibody inhibits one or more TREM2        activities, DAP12 activities, or both.        130. The isolated antibody of embodiment 129, wherein the one or        more TREM2 activities, DAP12 activities, or both comprise        decreasing activity of one or more TREM2-dependent genes.        131. The isolated antibody of embodiment 130, wherein the one or        more TREM2-dependent genes comprise one or more nuclear factor        of activated T-cells (NFAT) transcription factors.        132. The isolated antibody of any one of embodiments 129-131,        wherein the one or more TREM2 activities, DAP12 activities, or        both comprise decreasing the survival of macrophages, microglial        cells, M1 macrophages, M1 microglial cells, M2 macrophages, M2        microglial cells, osteoclasts, Langerhans cells of skin, Kupffer        cells, and/or dendritic cells.        133. The isolated antibody of any one of embodiments 125-132,        wherein the isolated antibody inhibits interaction between TREM2        and one or more TREM2 ligands, inhibits TREM2 signal        transduction, or both.        134. The isolated antibody of any one of embodiments 124-133,        wherein the antibody is incapable of binding an Fc-gamma        receptor (FcγR).        135. The isolated antibody of embodiment 134, wherein the        antibody has an IgG1 isotype.        136. The isolated antibody of embodiment 135, wherein the        antibody comprises a human IgG1 constant region.        137. The isolated antibody of embodiment 136, wherein the human        IgG1 constant region comprises an Fc region.        138. The isolated antibody of embodiment 137, wherein the Fc        region comprises one or more modifications.        139. The isolated antibody of embodiment 138, wherein the Fc        region comprises one or more amino acid substitutions.        140. The isolated antibody of embodiment 139, wherein the one or        more amino acid substitutions in the Fc region are at a residue        position selected from the group consisting of N297A, N297Q,        D265A, L234A, L235A, C226S, C229S, P238S, E233P, L234V, P238A,        A327Q, A327G, P329A, K322A, L234F, L235E, P331S, T394D, A330L,        M252Y, S254T, T256E, and any combination thereof, wherein the        numbering of the residues is according to EU numbering.        141. The isolated antibody of embodiment 140, wherein the Fc        region further comprises an amino acid deletion at a position        corresponding to glycine 236 according to EU numbering.        142. The isolated antibody of embodiment 135, wherein the        antibody comprises a mouse IgG1 constant region.        143. The isolated antibody of embodiment 134, wherein the        antibody has an IgG2 isotype.        144. The isolated antibody of embodiment 143, wherein the        antibody comprises a human IgG2 constant region.        145. The isolated antibody of embodiment 144, wherein the human        IgG2 constant region comprises an Fc region.        146. The isolated antibody of embodiment 145, wherein the Fc        region comprises one or more modifications.        147. The isolated antibody of embodiment 146, wherein the Fc        region comprises one or more amino acid substitutions.        148. The isolated antibody of embodiment 147, wherein the one or        more amino acid substitutions in the Fc region are at a residue        position selected from the group consisting of V234A, G237A,        H268E, V309L, N297A, N297Q, A330S, P331 S, C232S, C233S, M252Y,        S254T, T256E, and any combination thereof, wherein the numbering        of the residues is according to EU numbering.        149. The isolated antibody of embodiment 134, wherein the        antibody has an IgG4 isotype.        150. The isolated antibody of embodiment 149, wherein the        antibody comprises a human IgG4 constant region.        151. The isolated antibody of embodiment 150, wherein the human        IgG4 constant region comprises an Fc region.        152. The isolated antibody of embodiment 151, wherein the Fc        region comprises one or more modifications.        153. The isolated antibody of embodiment 152, wherein the Fc        region comprises one or more amino acid substitutions.        154. The isolated antibody of embodiment 153, wherein the one or        more amino acid substitutions in the Fc region are at a residue        position selected from the group consisting of E233P, F234V,        L235A, G237A, E318A, S228P, L236E, S241P, L248E, T394D, M252Y,        S254T, T256E, N297A, N297Q, and any combination thereof, wherein        the numbering of the residues is according to EU numbering.        155. The isolated antibody of any one of embodiments 124-154,        wherein the isolated antibody is an antibody fragment that binds        to one or more human proteins selected from the group consisting        of human TREM2, a naturally occurring variant of human TREM2,        human DAP12, and naturally occurring variant of human DAP12.        156. The isolated antibody of embodiment 155, wherein the        fragment is an Fab, Fab′, Fab′-SH, F(ab′)2, Fv or scFv fragment.        157. The isolated antibody of embodiment 106, embodiment 140, or        embodiment 141, wherein the Fc region further comprises one or        more additional amino acid substitutions at a position selected        from the group consisting of A330L, L234F; L235E, P331 S, and        any combination thereof, wherein the numbering of the residues        is according to EU numbering.        158. The isolated antibody of any one of embodiments 95-157,        wherein the Fc region further comprises one or more additional        amino acid substitutions at a position selected from the group        consisting of M252Y, S254T, T256E, and any combination thereof,        wherein the numbering of the residues is according to EU        numbering.        159. The isolated antibody of embodiment 118 or embodiment 154,        wherein the Fc region further comprises a S228P amino acid        substitution according to EU numbering.        160. The isolated antibody of any one of embodiments 44-159,        wherein the isolated antibody competes for binding of TREM2 with        one or more TREM2 ligands.        161. The isolated antibody of embodiment 160, wherein the one or        more TREM2 ligands are selected from the group consisting of E.        coli cells, apoptotic cells, nucleic acids, anionic lipids,        zwitterionic lipids, negatively charged phospholipids,        phosphatidylserine, sulfatides, phosphatidylcholin,        sphingomyelin, membrane phospholipids, lipidated proteins,        proteolipids, lipidated peptides, and lipidated amyloid beta        peptide.        162. The isolated antibody of any one of embodiments 44-161,        wherein the isolated antibody is a human antibody, a humanized        antibody, a bispecific antibody, a multivalent antibody, or a        chimeric antibody.        163. The isolated antibody of any one of embodiments 44-162,        wherein the isolated antibody is a bispecific antibody        recognizing a first antigen and a second antigen.        164. The isolated antibody of embodiment 163, wherein the first        antigen is human TREM2 or a naturally occurring variant thereof,        or human DAP12 or a naturally occurring variant thereof, and the        second antigen is a disease-causing protein selected from the        group consisting of amyloid beta or fragments thereof, Tau,        IAPP, alpha-synuclein, TDP-43, FUS protein, prion protein,        PrPSc, huntingtin, calcitonin, superoxide dismutase, ataxin,        Lewy body, atrial natriuretic factor, islet amyloid polypeptide,        insulin, apolipoprotein AI, serum amyloid A, medin, prolactin,        transthyretin, lysozyme, beta 2 microglobulin, gelsolin,        keratoepithelin, cystatin, immunoglobulin light chain AL, S-IBM        protein, Repeat-associated non-ATG (RAN) translation products,        DiPeptide repeat (DPR) peptides, glycine-alanine (GA) repeat        peptides, glycine-proline (GP) repeat peptides, glycine-arginine        (GR) repeat peptides, proline-alanine (PA) repeat peptides, and        proline-arginine (PR) repeat peptides; or a blood brain barrier        targeting protein selected from the group consisting of:        trasnferin receptor, insulin receptor, insulin like growth        factor receptor, LRP-1, and LRP1.        165. The isolated antibody of any one of embodiments 44-161,        wherein the isolated antibody is an antibody fragment that binds        to one or more human proteins selected from the group consisting        of human TREM2, a naturally occurring variant of human TREM2,        human DAP12, and naturally occurring variant of human DAP12; and        wherein the antibody is used in combination with one or more        antibodies that specifically bind a disease-causing protein        selected from the group consisting of: amyloid beta or fragments        thereof, Tau, IAPP, alpha-synuclein, TDP-43, FUS protein, prion        protein, PrPSc, huntingtin, calcitonin, superoxide dismutase,        ataxin, Lewy body, atrial natriuretic factor, islet amyloid        polypeptide, insulin, apolipoprotein AI, serum amyloid A, medin,        prolactin, transthyretin, lysozyme, beta 2 microglobulin,        gelsolin, keratoepithelin, cystatin, immunoglobulin light chain        AL, S-IBM protein, Repeat-associated non-ATG (RAN) translation        products, DiPeptide repeat (DPR) peptides, glycine-alanine (GA)        repeat peptides, glycine-proline (GP) repeat peptides,        glycine-arginine (GR) repeat peptides, proline-alanine (PA)        repeat peptides, and proline-arginine (PR) repeat peptides, and        any combination thereof.        166. The isolated antibody of any one of embodiments 44-165,        wherein the antibody is a monoclonal antibody.        167. The isolated antibody of any one of embodiments 44-166,        wherein the isolated antibody binds to a TREM2 protein, and        wherein the isolated antibody binds to one or more amino acids        within amino acid residues selected from the group consisting        of:    -   i. amino acid residues 29-112 of SEQ ID NO: 1, or amino acid        residues on a TREM2 protein corresponding to amino acid residues        29-112 of SEQ ID NO: 1;    -   ii. amino acid residues 29-41 of SEQ ID NO: 1, or amino acid        residues on a TREM2 protein corresponding to amino acid residues        29-41 of SEQ ID NO: 1;    -   iii. amino acid residues 40-44 of SEQ ID NO: 1, or amino acid        residues on a TREM2 protein corresponding to amino acid residues        40-44 of SEQ ID NO: 1;    -   iv. amino acid residues 47-69 of SEQ ID NO: 1, or amino acid        residues on a TREM2 protein corresponding to amino acid residues        47-69 of SEQ ID NO: 1;    -   v. amino acid residues 67-76 of SEQ ID NO: 1, or amino acid        residues on a TREM2 protein corresponding to amino acid residues        67-76 of SEQ ID NO: 1;    -   vi. amino acid residues 76-86 of SEQ ID NO: 1, or amino acid        residues on a TREM2 protein corresponding to amino acid residues        76-86 of SEQ ID NO: 1;    -   vii. amino acid residues 91-100 of SEQ ID NO: 1, or amino acid        residues on a TREM2 protein corresponding to amino acid residues        91-100 of SEQ ID NO: 1;    -   viii. amino acid residues 99-115 of SEQ ID NO: 1, or amino acid        residues on a TREM2 protein corresponding to amino acid residues        99-115 of SEQ ID NO: 1;    -   ix. amino acid residues 104-112 of SEQ ID NO: 1, or amino acid        residues on a TREM2 protein corresponding to amino acid residues        104-112 of SEQ ID NO: 1; and    -   x. amino acid residues 114-118 of SEQ ID NO: 1, or amino acid        residues on a TREM2 protein corresponding to amino acid residues        114-118 of SEQ ID NO: 1.        168. The isolated antibody of embodiment 167, wherein the        isolated antibody binds to one or more amino acids within amino        acid residues 43-50 of SEQ ID NO: 1, or amino acid residues on a        TREM2 protein corresponding to amino acid residues 43-50 of SEQ        ID NO: 1.        169. The isolated antibody of embodiment 167, wherein the        isolated antibody binds to one or more amino acids within amino        acid residues 49-57 of SEQ ID NO: 1, or amino acid residues on a        TREM2 protein corresponding to amino acid residues 49-57 of SEQ        ID NO: 1.        170. The isolated antibody of any one of embodiments 44-167,        wherein the isolated antibody binds to an epitope comprising one        or more amino acids within amino acid residues 43-50 of SEQ ID        NO: 1.        171. The isolated antibody of any one of embodiments 44-167,        wherein the isolated antibody binds to an epitope comprising one        or more amino acids within amino acid residues 43-50 of SEQ ID        NO: 1.        172. The isolated antibody of any one of embodiments 44-167,        wherein the isolated antibody binds to an epitope comprising one        or more amino acid residues selected from the group consisting        of:    -   i. amino acid residue Arg47 or Asp87 of SEQ ID NO: 1;    -   ii. amino acid residues 40-44 of SEQ ID NO: 1;    -   iii. amino acid residues 67-76 of SEQ ID NO: 1; and    -   iv. amino acid residues 114-118 of SEQ ID NO: 1.        173. The isolated antibody of any one of embodiments 44-167,        wherein the isolated antibody binds to one or more amino acids        within amino acid residues 22-40 of SEQ ID NO: 2, or amino acid        residues on a DAP12 protein corresponding to amino acid residues        22-40 of SEQ ID NO: 2.        174. The isolated antibody of any one of embodiments 44-167,        wherein the isolated antibody is a bispecific antibody that        binds to one or more amino acids selected from the group        consisting of:    -   i. one or more amino acid residues of SEQ ID NO: 1, or amino        acid residues on a TREM2 protein corresponding to amino acid        residues of SEQ ID NO: 1; and    -   ii. one or more amino acid residues of SEQ ID NO: 2, or amino        acid residues on a DAP12 protein corresponding to amino acid        residues of SEQ ID NO: 2.        175. The isolated antibody of any one of embodiments 44-174,        wherein the isolated antibody comprises a heavy chain variable        domain and a light chain variable domain, wherein the heavy        chain variable domain comprises the HVR-H1, HVR-H2, and/or        HVR-H3 of the monoclonal antibody Ab52; and/or wherein the light        chain variable domain comprises the HVR-L1, HVR-L2, and/or        HVR-L3 of the monoclonal antibody Ab52.        176. The isolated antibody of embodiment 175, wherein the HVR-H1        comprises the amino acid sequence of SEQ ID NO:398.        177. The isolated antibody of embodiment 175 or embodiment 176,        wherein the HVR-H2 comprises the amino acid sequence of SEQ ID        NO:399.        178. The isolated antibody of any one of embodiments 175-177,        wherein the HVR-H3 comprises the amino acid sequence of SEQ ID        NO:400.        179. The isolated antibody of any one of embodiments 175-178,        wherein the HVR-L1 comprises the amino acid sequence of SEQ ID        NO:401.        180. The isolated antibody of any one of embodiments 175-179,        wherein the HVR-L2 comprises the amino acid sequence of SEQ ID        NO:402.        181. The isolated antibody of any one of embodiments 175-180,        wherein the HVR-L3 comprises the amino acid sequence of SEQ ID        NO:403.        182. The isolated antibody of any one of embodiments 44-174,        wherein the isolated antibody comprises a heavy chain variable        domain and a light chain variable domain, wherein the heavy        chain variable domain comprises    -   (a) an HVR-H1 comprising the amino acid sequence of SEQ ID        NO:398, or an amino acid sequence with at least about 95%        homology to the amino acid sequence of SEQ ID NO:398;    -   (b) an HVR-H2 comprising the amino acid sequence of SEQ ID        NO:399, or an amino acid sequence with at least about 95%        homology to the amino acid sequence of SEQ ID NO:399; and    -   (c) an HVR-H3 comprising the amino acid sequence of SEQ ID        NO:400, or an amino acid sequence with at least about 95%        homology to the amino acid sequence of SEQ ID NO:400; and/or        wherein the light chain variable domain comprises:    -   (a) an HVR-L1 comprising the amino acid sequence of SEQ ID        NO:401, or an amino acid sequence with at least about 95%        homology to the amino acid sequence of SEQ ID NO:401;    -   (b) an HVR-L2 comprising the amino acid sequence of SEQ ID        NO:402, or an amino acid sequence with at least about 95%        homology to the amino acid sequence of SEQ ID NO:402; and    -   (c) an HVR-L3 comprising the amino acid sequence of SEQ ID        NO:403, or an amino acid sequence with at least about 95%        homology to the amino acid sequence of SEQ ID NO:403.        183. The isolated antibody of any one of embodiments 44-174,        wherein the isolated antibody comprises a heavy chain variable        domain and a light chain variable domain, wherein the heavy        chain variable domain comprises the HVR-H1, HVR-H2, and/or        HVR-H3 of the monoclonal antibody Ab21; and/or wherein the light        chain variable domain comprises the HVR-L1, HVR-L2, and/or        HVR-L3 of the monoclonal antibody Ab21.        184. The isolated antibody of embodiment 183, wherein the HVR-H1        comprises the amino acid sequence of SEQ ID NO:404.        185. The isolated antibody of embodiment 183 or 184, wherein the        HVR-H2 comprises the amino acid sequence of SEQ ID NO:405.        186. The isolated antibody of any one of embodiments 183-185,        wherein the HVR-H3 comprises the amino acid sequence of SEQ ID        NO:406.        187. The isolated antibody of any one of embodiments 183-186,        wherein the HVR-L comprises the amino acid sequence of SEQ ID        NO:407.        188. The isolated antibody of any one of embodiments 183-187,        wherein the HVR-L2 comprises the amino acid sequence of SEQ ID        NO:408.        189. The isolated antibody of any one of embodiments 183-188,        wherein the HVR-L3 comprises the amino acid sequence of SEQ ID        NO:409.        190. The isolated antibody of any one of embodiments 44-174,        wherein the isolated antibody comprises a heavy chain variable        domain and a light chain variable domain, wherein the heavy        chain variable domain comprises    -   (a) an HVR-H1 comprising the amino acid sequence of SEQ ID        NO:404, or an amino acid sequence with at least about 95%        homology to the amino acid sequence of SEQ ID NO:404;    -   (b) an HVR-H2 comprising the amino acid sequence of SEQ ID        NO:405, or an amino acid sequence with at least about 95%        homology to the amino acid sequence of SEQ ID NO:405; and    -   (c) an HVR-H3 comprising the amino acid sequence of SEQ ID        NO:406, or an amino acid sequence with at least about 95%        homology to the amino acid sequence of SEQ ID NO:406; and/or        wherein the light chain variable domain comprises:    -   (a) an HVR-L1 comprising the amino acid sequence of SEQ ID        NO:407, or an amino acid sequence with at least about 95%        homology to the amino acid sequence of SEQ ID NO:407;    -   (b) an HVR-L2 comprising the amino acid sequence of SEQ ID        NO:408, or an amino acid sequence with at least about 95%        homology to the amino acid sequence of SEQ ID NO:408; and    -   (c) an HVR-L3 comprising the amino acid sequence of SEQ ID        NO:409, or an amino acid sequence with at least about 95%        homology to the amino acid sequence of SEQ ID NO:409.        191. An isolated anti-human TREM2 antibody, wherein the isolated        antibody comprises a heavy chain variable domain and a light        chain variable domain, wherein the heavy chain variable domain        comprises the HVR-H1, HVR-H2, and/or HVR-H3 of the monoclonal        antibody Ab52; and/or wherein the light chain variable domain        comprises the HVR-L1, HVR-L2, and/or HVR-L3 of the monoclonal        antibody Ab52.        192. The isolated antibody of embodiment 191, wherein the HVR-H1        comprises the amino acid sequence of SEQ ID NO:398.        193. The isolated antibody of embodiment 191 or embodiment 192,        wherein the HVR-H2 comprises the amino acid sequence of SEQ ID        NO:399.        194. The isolated antibody of any one of embodiments 191-193,        wherein the HVR-H3 comprises the amino acid sequence of SEQ ID        NO:400.        195. The isolated antibody of any one of embodiments 191-194,        wherein the HVR-L1 comprises the amino acid sequence of SEQ ID        NO:401.        196. The isolated antibody of any one of embodiments 191-195,        wherein the HVR-L2 comprises the amino acid sequence of SEQ ID        NO:402.        197. The isolated antibody of any one of embodiments 191-196,        wherein the HVR-L3 comprises the amino acid sequence of SEQ ID        NO:403.        198. The isolated antibody of embodiment 191, wherein the        isolated antibody comprises a heavy chain variable domain and a        light chain variable domain, wherein the heavy chain variable        domain comprises an HVR-H1 comprising the amino acid sequence of        SEQ ID NO:398, an HVR-H2 comprising the amino acid sequence of        SEQ ID NO:399, and an HVR-H3 comprising the amino acid sequence        of SEQ ID NO:400, and/or wherein the light chain variable domain        comprises an HVR-L1 comprising the amino acid sequence of SEQ ID        NO:401, an HVR-L2 comprising the amino acid sequence of SEQ ID        NO:402, and an HVR-L3 comprising the amino acid sequence of SEQ        ID NO:403.        199. An isolated anti-human TREM2 antibody, wherein the isolated        antibody comprises a heavy chain variable domain and a light        chain variable domain, wherein the heavy chain variable domain        comprises    -   (a) an HVR-H1 comprising the amino acid sequence of SEQ ID        NO:398, or an amino acid sequence with at least about 95%        homology to the amino acid sequence of SEQ ID NO:398;    -   (b) an HVR-H2 comprising the amino acid sequence of SEQ ID        NO:399, or an amino acid sequence with at least about 95%        homology to the amino acid sequence of SEQ ID NO:399; and    -   (c) an HVR-H3 comprising the amino acid sequence of SEQ ID        NO:400, or an amino acid sequence with at least about 95%        homology to the amino acid sequence of SEQ ID NO:400; and/or        wherein the light chain variable domain comprises:    -   (a) an HVR-L1 comprising the amino acid sequence of SEQ ID        NO:401, or an amino acid sequence with at least about 95%        homology to the amino acid sequence of SEQ ID NO:401;    -   (b) an HVR-L2 comprising the amino acid sequence of SEQ ID        NO:402, or an amino acid sequence with at least about 95%        homology to the amino acid sequence of SEQ ID NO:402; and    -   (c) an HVR-L3 comprising the amino acid sequence of SEQ ID        NO:403, or an amino acid sequence with at least about 95%        homology to the amino acid sequence of SEQ ID NO:403.        200. An isolated anti-human TREM2 antibody, wherein the isolated        antibody comprises a heavy chain variable domain and a light        chain variable domain, wherein the heavy chain variable domain        comprises the HVR-H1, HVR-H2, and/or HVR-H3 of the monoclonal        antibody Ab21; and/or wherein the light chain variable domain        comprises the HVR-L1, HVR-L2, and/or HVR-L3 of the monoclonal        antibody Ab21.        201. The isolated antibody of embodiment 200, wherein the HVR-H1        comprises the amino acid sequence of SEQ ID NO:404.        202. The isolated antibody of embodiment 200 or embodiment 201,        wherein the HVR-H2 comprises the amino acid sequence of SEQ ID        NO:405.        203. The isolated antibody of any one of embodiments 200-202,        wherein the HVR-H3 comprises the amino acid sequence of SEQ ID        NO:406.        204. The isolated antibody of any one of embodiments 200-203,        wherein the HVR-L1 comprises the amino acid sequence of SEQ ID        NO:407.        205. The isolated antibody of any one of embodiments 200-204,        wherein the HVR-L2 comprises the amino acid sequence of SEQ ID        NO:408.        206. The isolated antibody of any one of embodiments 200-205,        wherein the HVR-L3 comprises the amino acid sequence of SEQ ID        NO:409.        207. The isolated antibody of embodiment 200, wherein the        isolated antibody comprises a heavy chain variable domain and a        light chain variable domain, wherein the heavy chain variable        domain comprises an HVR-H1 comprising the amino acid sequence of        SEQ ID NO:404, an HVR-H2 comprising the amino acid sequence of        SEQ ID NO:405, and an HVR-H3 comprising the amino acid sequence        of SEQ ID NO:406, and/or wherein the light chain variable domain        comprises an HVR-L comprising the amino acid sequence of SEQ ID        NO:407, an HVR-L2 comprising the amino acid sequence of SEQ ID        NO:408, and an HVR-L3 comprising the amino acid sequence of SEQ        ID NO:409.        208. An isolated anti-human TREM2 antibody, wherein the isolated        antibody comprises a heavy chain variable domain and a light        chain variable domain, wherein the heavy chain variable domain        comprises    -   (a) an HVR-H1 comprising the amino acid sequence of SEQ ID        NO:404, or an amino acid sequence with at least about 95%        homology to the amino acid sequence of SEQ ID NO:404;    -   (b) an HVR-H2 comprising the amino acid sequence of SEQ ID        NO:405, or an amino acid sequence with at least about 95%        homology to the amino acid sequence of SEQ ID NO:405; and    -   (c) an HVR-H3 comprising the amino acid sequence of SEQ ID        NO:406, or an amino acid sequence with at least about 95%        homology to the amino acid sequence of SEQ ID NO:406; and/or        wherein the light chain variable domain comprises:    -   (a) an HVR-L1 comprising the amino acid sequence of SEQ ID        NO:407, or an amino acid sequence with at least about 95%        homology to the amino acid sequence of SEQ ID NO:407;    -   (b) an HVR-L2 comprising the amino acid sequence of SEQ ID        NO:408, or an amino acid sequence with at least about 95%        homology to the amino acid sequence of SEQ ID NO:408; and    -   (c) an HVR-L3 comprising the amino acid sequence of SEQ ID        NO:409, or an amino acid sequence with at least about 95%        homology to the amino acid sequence of SEQ ID NO:409.        209. An isolated anti-human TREM2 antibody which binds        essentially the same TREM2 epitope as the antibody Ab52.        210. An isolated anti-human TREM2 antibody which binds        essentially the same TREM2 epitope as the antibody Ab21.        211. The isolated antibody of any one of embodiments 191-210,        wherein the antibody is an agonist antibody, and wherein the        antibody induces one or more TREM2 activities, DAP12 activities,        or both.        212. The isolated antibody of embodiment 211, wherein the        isolated antibody induces or retains TREM2 clustering, DAP12        clustering, or both on a cell surface.        213. The isolated antibody of embodiment 211 or embodiment 212,        wherein the one or more TREM2 activities, DAP12 activities, or        both are selected from the group consisting of TREM2 binding to        DAP12; DAP12 binding to TREM2; TREM2 phosphorylation, DAP12        phosphorylation; PI3K activation; increased expression of one or        more anti-inflammatory mediators; reduced expression of one or        more pro-inflammatory mediators; reduced expression of TNF-α,        IL-6, or both; extracellular signal-regulated kinase (ERK)        phosphorylation; increased expression of C-C chemokine receptor        7 (CCR7); induction of microglial cell chemotaxis toward CCL19        and CCL21 expressing cells; an increase, normalization, or both        of the ability of bone marrow-derived dendritic cells to induce        antigen-specific T-cell proliferation; induction of osteoclast        production, increased rate of osteoclastogenesis, or both;        increasing the survival and/or function of one or more of        dendritic cells, macrophages, microglial cells, M1 macrophages        and/or microglial cells, activated M1 macrophages and/or        microglial cells, M2 macrophages and/or microglial cells,        osteoclasts, Langerhans cells of skin, and Kupffer cells;        induction of one or more types of clearance selected from the        group consisting of apoptotic neuron clearance, nerve tissue        debris clearance, non-nerve tissue debris clearance, bacteria or        other foreign body clearance, disease-causing protein clearance,        disease-causing peptide clearance, and disease-causing nucleic        acid clearance; induction of phagocytosis of one or more of        apoptotic neurons, nerve tissue debris, non-nerve tissue debris,        bacteria, other foreign bodies, disease-causing proteins,        disease-causing peptides, or disease-causing nucleic acids;        normalization of disrupted TREM2/DAP12-dependent gene        expression; recruitment of Syk, ZAP70, or both to the        TREM2/DAP12 complex; Syk phosphorylation; increased expression        of CD83 and/or CD86 on dendritic cells; reduced secretion of one        or more inflammatory cytokines; reduced expression of one or        more inflammatory receptors; increasing phagocytosis by        macrophages, dendritic cells, monocytes, and/or microglia under        conditions of reduced levels of MCSF; decreasing phagocytosis by        macrophages, dendritic cells, monocytes, and/or microglia in the        presence of normal levels of MCSF; increasing activity of one or        more TREM2-dependent genes; and any combination thereof.        214. The isolated antibody of any one of embodiments 211-213,        wherein the antibody is of the IgG class the IgM class, or the        IgA class.        215. The isolated antibody of embodiment 214, wherein the        antibody is of the IgG class and has an IgG1, IgG2, IgG3, or        IgG4 isotype.        216. The isolated antibody of embodiment 215, wherein the        antibody has an IgG2 isotype.        217. The isolated antibody of embodiment 216, wherein the        antibody comprises a human IgG2 constant region.        218. The isolated antibody of embodiment 217, wherein the human        IgG2 constant region comprises an Fc region.        219. The isolated antibody of any one of embodiments 216-218,        wherein the antibody induces the one or more TREM2 activities,        DAP12 activities, or both independently of binding to an Fc        receptor.        220. The isolated antibody of any one of embodiments 216-218,        wherein the antibody binds an inhibitory Fc receptor.        221. The isolated antibody of embodiment 220, wherein the        inhibitory Fc receptor is inhibitory Fc-gamma receptor IIB        (FcγIIB).        222. The isolated antibody of embodiment 220 or embodiment 221,        wherein the Fc region comprises one or more modifications.        223. The isolated antibody of embodiment 222, wherein the Fc        region comprises one or more amino acid substitutions.        224. The isolated antibody of embodiment 223, wherein the one or        more amino acid substitutions in the Fc region are at a residue        position selected from the group consisting of V234A, G237A,        H268Q, V309L, A330S, P331S, C232S, C233S, S267E, L328F, M252Y,        S254T, T256E, and any combination thereof, wherein the numbering        of the residues is according to EU numbering.        225. The isolated antibody of embodiment 217, wherein the human        IgG2 constant region comprises a light chain constant region        comprising a C214S amino acid substitution, wherein the        numbering of the residues is according to EU numbering.        226. The isolated antibody of embodiment 215, wherein the        antibody has an IgG1 isotype.        227. The isolated antibody of embodiment 226, wherein the        antibody comprises a human IgG1 constant region.        228. The isolated antibody of embodiment 227, wherein the human        IgG1 constant region comprises an Fc region.        229. The isolated antibody of any one of embodiments 226-228,        wherein the antibody binds an inhibitory Fc receptor.        230. The isolated antibody of embodiment 229, wherein the        inhibitory Fc receptor is inhibitory Fc-gamma receptor IIB        (FcγRIIB).        231. The isolated antibody of any one of embodiments 228-230,        wherein the Fc region comprises one or more modifications.        232. The isolated antibody of embodiment 231, wherein the Fc        region comprises one or more amino acid substitutions.        233. The isolated antibody of embodiment 232, wherein the one or        more amino acid substitutions in the Fc region are at a residue        position selected from the group consisting of N297A, D265A,        L234A, L235A, G237A, C226S, C229S, E233P, L234V, L234F, L235E,        P331S, S267E, L328F, A330L, M252Y, S254T, T256E, and any        combination thereof, wherein the numbering of the residues is        according to EU numbering.        234. The isolated antibody of any one of embodiments 228-230,        wherein the antibody comprises an IgG2 isotype heavy chain        constant domain 1(CH1) and hinge region.        235. The isolated antibody of embodiment 234, wherein the IgG2        isotype CH1 and hinge region comprise the amino acid sequence of        ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGVHTFPAVLQSS        GLYSLSSVVT VPSSNFGTQT YTCNVDHKPS NTKVDKTVERKCCVECPPCP (SEQ ID        NO: 397).        236. The isolated antibody of embodiment 234 or embodiment 235,        wherein the antibody Fc region comprises a S267E amino acid        substitution, a L328F amino acid substitution, or both, and/or a        N297A or N297Q amino acid substitution, wherein the numbering of        the residues is according to EU numbering.        237. The isolated antibody of embodiment 226, wherein the        antibody comprises a mouse IgG1 constant region.        238. The isolated antibody of embodiment 215, wherein the        antibody has an IgG4 isotype.        239. The isolated antibody of embodiment 238, wherein the        antibody comprises a human IgG4 constant region.        240. The isolated antibody of embodiment 239, wherein the human        IgG4 constant region comprises an Fc region.        241. The isolated antibody of any one of embodiments 238-240,        wherein the antibody binds an inhibitory Fc receptor.        242. The isolated antibody of embodiment 241, wherein the        inhibitory Fc receptor is inhibitory Fc-gamma receptor IIB        (FcγIIB).        243. The isolated antibody of any one of embodiments 240-242,        wherein the Fc region comprises one or more modifications.        244. The isolated antibody of embodiment 243, wherein the Fc        region comprises one or more amino acid substitutions.        245. The isolated antibody of embodiment 244, wherein the one or        more amino acid substitutions in the Fc region are at a residue        position selected from the group consisting of L235A, G237A,        S228P, L236E, S267E, E318A, L328F, M252Y, S254T, T256E, and any        combination thereof, wherein the numbering of the residues is        according to EU numbering.        246. The isolated antibody of embodiment 214, wherein the        antibody has a hybrid IgG2/4 isotype.        247. The isolated antibody of embodiment 246, wherein the        antibody comprises an amino acid sequence comprising amino acids        118 to 260 of human IgG2 and amino acids 261 to 447 of human        IgG4, wherein the numbering of the residues is according to EU        numbering.        248. The isolated antibody of embodiment 239, wherein the        antibody comprises a mouse IgG4 constant region.        249. The isolated antibody of any one of embodiments 211-248,        wherein the isolated antibody is an antibody fragment that binds        to one or more human proteins selected from the group consisting        of human TREM2, a naturally occurring variant of human TREM2,        human DAP12, and naturally occurring variant of human DAP12, and        wherein the antibody fragment is cross-linked to a second        antibody fragment that binds to one or more human proteins        selected from the group consisting of human TREM2, a naturally        occurring variant of human TREM2, human DAP12, and naturally        occurring variant of human DAP12.        250. The isolated antibody of embodiment 249, wherein the        fragment is an Fab, Fab′, Fab′-SH, F(ab′)2, Fv or scFv fragment.        251. The isolated antibody of any one of embodiments 191-210,        wherein the isolated antibody is an inert antibody.        252. The isolated antibody of any one of embodiments 191-210,        wherein the isolated antibody is an antagonist antibody.        253. The isolated antibody of embodiment 252, wherein the        isolated antibody inhibits one or more TREM2 activities.        254. The isolated antibody of embodiment 253, wherein the one or        more TREM2 activities are selected from the group consisting of        decreasing activity of one or more TREM2-dependent genes;        decreasing activity of one or more nuclear factor of activated        T-cells (NFAT) transcription factors; decreasing the survival of        macrophages, microglial cells, monocytes, osteoclasts,        Langerhans cells of skin, Kupffer cells, and/or dendritic cells;        and any combination thereof.        255. The isolated antibody of any one of embodiments 252-254,        wherein the isolated antibody inhibits interaction between TREM2        and one or more TREM2 ligands, inhibits TREM2 signal        transduction, or both.        256. The isolated antibody of any one of embodiments 251-255,        wherein the antibody is incapable of binding an Fc-gamma        receptor (FcγR).        257. The isolated antibody of embodiment 256, wherein the        antibody has an IgG1 isotype.        258. The isolated antibody of embodiment 257, wherein the        antibody comprises a human IgG1 constant region.        259. The isolated antibody of embodiment 258, wherein the human        IgG1 constant region comprises an Fc region.        260. The isolated antibody of embodiment 259, wherein the Fc        region comprises one or more modifications.        261. The isolated antibody of embodiment 260, wherein the Fe        region comprises one or more amino acid substitutions.        262. The isolated antibody of embodiment 261, wherein the one or        more amino acid substitutions in the Fc region are at a residue        position selected from the group consisting of N297A, N297Q,        D265A, L234A, L235A, C226S, C229S, P238S, E233P, L234V, P238A,        A327Q, A327G, P329A, K322A, L234F, L235E, P331S, T394D, A330L,        M252Y, S254T, T256E, and any combination thereof, wherein the        numbering of the residues is according to EU numbering.        263. The isolated antibody of embodiment 262, wherein the Fc        region further comprises an amino acid deletion at a position        corresponding to glycine 236 according to EU numbering.        264. The isolated antibody of embodiment 257, wherein the        antibody comprises a mouse IgG1 constant region.        265. The isolated antibody of embodiment 256, wherein the        antibody has an IgG2 isotype.        266. The isolated antibody of embodiment 264, wherein the        antibody comprises a human IgG2 constant region.        267. The isolated antibody of embodiment 266, wherein the human        IgG2 constant region comprises an Fc region.        268. The isolated antibody of embodiment 267, wherein the Fc        region comprises one or more modifications.        269. The isolated antibody of embodiment 268, wherein the Fc        region comprises one or more amino acid substitutions.        270. The isolated antibody of embodiment 269, wherein the one or        more amino acid substitutions in the Fc region are at a residue        position selected from the group consisting of V234A, G237A,        H268E, V309L, N297A, N297Q, A330S, P331S, C232S, C233S, M252Y,        S254T, T256E, and any combination thereof, wherein the numbering        of the residues is according to EU numbering.        271. The isolated antibody of embodiment 256, wherein the        antibody has an IgG4 isotype.        272. The isolated antibody of embodiment 271, wherein the        antibody comprises a human IgG4 constant region.        273. The isolated antibody of embodiment 272, wherein the human        IgG4 constant region comprises an Fc region.        274. The isolated antibody of embodiment 273, wherein the Fc        region comprises one or more modifications.        275. The isolated antibody of embodiment 274, wherein the Fc        region comprises one or more amino acid substitutions.        276. The isolated antibody of embodiment 275, wherein the one or        more amino acid substitutions in the Fc region are at a residue        position selected from the group consisting of E233P, F234V,        L235A, G237A, E318A, S228P, L236E, S241P, L248E, T394D, M252Y,        S254T, T256E, N297A, N297Q, and any combination thereof, wherein        the numbering of the residues is according to EU numbering.        277. The isolated antibody of any one of embodiments 208-276,        wherein the isolated antibody is an antibody fragment that binds        to one or more human proteins selected from the group consisting        of human TREM2, a naturally occurring variant of human TREM2,        human DAP12, and naturally occurring variant of human DAP12.        278. The isolated antibody of embodiment 277, wherein the        fragment is an Fab, Fab′, Fab′-SH, F(ab′)2, Fv or scFv fragment.        279. The isolated antibody of embodiment 233, embodiment 262, or        embodiment 263, wherein the Fc region further comprises one or        more additional amino acid substitutions at a position selected        from the group consisting of A330L, L234F; L235E, P331S, and any        combination thereof, wherein the numbering of the residues is        according to EU numbering.        280. The isolated antibody of any one of embodiments 223-279,        wherein the Fc region further comprises one or more additional        amino acid substitutions at a position selected from the group        consisting of M252Y, S254T, T256E, and any combination thereof,        wherein the numbering of the residues is according to EU        numbering.        281. The isolated antibody of embodiment 245 or embodiment 276,        wherein the Fc region further comprises a S228P amino acid        substitution according to EU numbering.        282. The isolated antibody of any one of embodiments 191-281,        wherein the antibody is a human antibody, a humanized antibody,        a bispecific antibody, a multivalent antibody, or a chimeric        antibody.        283. The isolated antibody of any one of embodiments 191-282,        wherein the antibody is a bispecific antibody recognizing a        first antigen and a second antigen.        284. The isolated antibody of any one of embodiments 191-283,        wherein the antibody is a monoclonal antibody.        285. The isolated antibody of any one of the preceding        embodiments, wherein the isolated antibody binds specifically to        both human TREM2 and mouse TREM2.        286. The isolated antibody of any one of the preceding        embodiments, wherein the isolated antibody has dissociation        constant (K_(D)) for human TREM2 and mouse TREM2 that ranges        from less than about 5.75 nM to less than about 0.09 nM.        287. The isolated antibody of any one of the preceding        embodiments, wherein the isolated antibody has dissociation        constant (K_(D)) for human TREM2-Fc fusion protein that ranges        from less than about 1.51 nM to less than about 0.35 nM.        288. The isolated antibody of any one of the preceding        embodiments, wherein the isolated antibody has dissociation        constant (K_(D)) for human monomeric TREM2 protein that ranges        from less than about 5.75 nM to less than about 1.15 nm.        289. The isolated antibody of any one of the preceding        embodiments, wherein the isolated antibody has dissociation        constant (K_(D)) for mouse TREM2-Fc fusion protein that ranges        from less than about 0.23 nM to less than about 0.09 nM.        290. An isolated nucleic acid encoding the antibody of any one        of the preceding embodiments.        291. A vector comprising the nucleic acid of embodiment 290.        292. A host cell comprising the vector of embodiment 291.        293. A method of producing an antibody, comprising culturing the        cell of embodiment 292 so that the antibody is produced.        294. The method of embodiment 293, further comprising recovering        the antibody produced by the cell.        295. A pharmaceutical composition comprising the antibody of any        one of embodiments 44-289 and a pharmaceutically acceptable        carrier.        296. A method of preventing, reducing risk, or treating an        individual having a disease, disorder, or injury selected from        the group consisting of dementia, frontotemporal dementia,        Alzheimer's disease, Nasu-Hakola disease, and multiple        sclerosis, comprising administering to the individual a        therapeutically effective amount of an isolated antibody that        binds to a TREM2 protein, a DAP12 protein, or both.        297. The method of embodiment 296, wherein the isolated antibody        is the isolated antibody of any one of embodiments 44-289.        298. The method of embodiment 296 or embodiment 297, wherein the        individual has a heterozygous variant of TREM2, wherein the        variant comprises one or more substitutions selected from the        group consisting of:    -   i. a glutamic acid to stop codon substitution in the nucleic        acid sequence encoding amino acid residue Glu14 of SEQ ID NO: 1;    -   ii. a glutamine to stop codon substitution in the nucleic acid        sequence encoding amino acid residue Gln33 of SEQ ID NO: 1;    -   iii. a tryptophan to stop codon substitution in the nucleic acid        sequence encoding amino acid residue Trp44 of SEQ ID NO: 1;    -   iv. an arginine to histidine amino acid substitution at an amino        acid corresponding to amino acid residue Arg47 of SEQ ID NO: 1;    -   v. a tryptophan to stop codon substitution in the nucleic acid        sequence encoding amino acid residue Trp78 of SEQ ID NO: 1;    -   vi. a valine to glycine amino acid substitution at an amino acid        corresponding to amino acid residue Val126 of SEQ ID NO: 1;    -   vii. an aspartic acid to glycine amino acid substitution at an        amino acid corresponding to amino acid residue Asp134 of SEQ ID        NO: 1; and    -   viii. a lysine to asparagine amino acid substitution at an amino        acid corresponding to amino acid residue Lys186 of SEQ ID NO: 1.        299. The method of embodiment 296, wherein the individual has a        heterozygous variant of TREM2, wherein the variant comprises a        guanine nucleotide deletion at a nucleotide corresponding to        nucleotide residue G313 of the nucleic acid sequence encoding        SEQ ID NO: 1; a guanine nucleotide deletion at a nucleotide        corresponding to nucleotide residue G267 of the nucleic acid        sequence encoding SEQ ID NO: 1; or both.        300. The method of embodiment 296, wherein the individual has a        heterozygous variant of DAP12, wherein the variant comprises one        or more variants selected from the group consisting of:    -   i. a methionine to threonine substitution at an amino acid        corresponding to amino acid residue Met1 of SEQ ID NO: 2;    -   ii. a glycine to arginine amino acid substitution at an amino        acid corresponding to amino acid residue Gly49 of SEQ ID NO: 2;    -   iii. a deletion within exons 1-4 of the nucleic acid sequence        encoding SEQ ID NO: 2;    -   iv. an insertion of 14 amino acid residues at exon 3 of the        nucleic acid sequence encoding SEQ ID NO: 2; and    -   v. a guanine nucleotide deletion at a nucleotide corresponding        to nucleotide residue G141 of the nucleic acid sequence encoding        SEQ ID NO: 2.        301. A method of inducing or promoting innate immune cell        survival an individual in need thereof, comprising administering        to the individual a therapeutically effective amount of an        isolated agonist antibody that binds to a TREM2 protein, a DAP12        protein, or both.        302. The method of embodiment 301 wherein the isolated agonist        antibody is the isolated antibody of any one of embodiments        44-123, 157-250, and 277-289.        303. An isolated agonist antibody that binds to a TREM2 protein,        wherein the antibody induces one or more TREM2 activities.        304. The isolated antibody of embodiment 303, wherein the TREM2        protein is a mammalian protein or a human protein.        305. The isolated antibody of embodiment 304, wherein the TREM2        protein is a wild-type protein.        306. The isolated antibody of embodiment 304, wherein the TREM2        protein is a naturally occurring variant.        307. The isolated antibody of any one of embodiments 303-306,        wherein the TREM2 protein is expressed on human dendritic cells,        human macrophages, human monocytes, human osteoclasts, human        Langerhans cells of skin, human Kupffer cells, and/or human        microglia.        308. The isolated antibody of any one of embodiments 303-307,        wherein the isolated antibody induces or retains TREM2        clustering on a cell surface.        309. The isolated antibody of any one of embodiments 303-308,        wherein the one or more TREM2 activities comprise TREM2 binding        to DAP12.        310. The isolated antibody of any one of embodiments 303-309,        wherein the one or more TREM2 activities comprise TREM2        autophosphorylation.        311. The isolated antibody of any one of embodiments 303-310,        wherein the one or more TREM2 activities comprise DAP12        phosphorylation.        312. The isolated antibody of embodiment 310 or embodiment 311,        wherein the TREM2 autophosphorylation, the DAP12        phosphorylation, or both is induced by one or more SRC family        tyrosine kinases.        313. The isolated antibody of embodiment 312, wherein the one or        more SRC family tyrosine kinases comprise a Syk kinase.        314. The isolated antibody of any one of embodiments 303-313,        wherein the one or more TREM2 activities comprise PI3K        activation.        315. The isolated antibody of any one of embodiments 303-314,        wherein the one or more TREM2 activities comprise increased        expression of one or more anti-inflammatory cytokines.        316. The isolated antibody of any one of embodiments 303-314,        wherein the one or more TREM2 activities comprise increased        expression of one or more cytokines selected from the group        consisting of IL-12p70, IL-6, and IL-10.        317. The isolated antibody of embodiment 315 or embodiment 316,        wherein the increased expression occurs in one or more cells        selected from the group consisting of macrophages, dendritic        cells, monocytes, osteoclasts, Langerhans cells of skin, Kupffer        cells, and microglial cells.        318. The isolated antibody of any one of embodiments 303-317,        wherein the one or more TREM2 activities comprise reduced        expression of one or more pro-inflammatory cytokines.        319. The isolated antibody of any one of embodiments 303-317,        wherein the one or more TREM2 activities comprise reduced        expression of one or more pro-inflammatory mediators selected        from the group consisting of IFN-a4, IFN-b, IL-1β, TNF-α, IL-10,        IL-6, IL-8, CRP, TGF-beta members of the chemokine protein        families, IL-20 family members, IL-33, LIF, IFN-gamma, OSM,        CNTF, TGF-beta, GM-CSF, IL-11, IL-12, IL-17, IL-18, and CRP.        320. The isolated antibody of any one of embodiments 303-318,        wherein the one or more TREM2 activities comprise reduced        expression of TNF-α, IL-6, or both.        321. The isolated antibody of embodiment 319 or embodiment 320,        wherein the reduced expression of the one or more        pro-inflammatory mediators occurs in one or more cells selected        from the group consisting of macrophages, dendritic cells,        monocytes, osteoclasts, Langerhans cells of skin, Kupffer cells,        and microglial cells.        322. The isolated antibody of any one of embodiments 303-320,        wherein the one or more TREM2 activities comprise extracellular        signal-regulated kinase (ERK) phosphorylation.        323. The isolated antibody of any one of embodiments 303-322,        wherein the one or more TREM2 activities comprise increased        expression of C-C chemokine receptor 7 (CCR7).        324. The isolated antibody of any one of embodiments 303-322,        wherein the one or more TREM2 activities comprise induction of        microglial cell chemotaxis toward CCL19 and CCL21 expressing        cells.        325. The isolated antibody of any one of embodiments 303-324,        wherein the one or more TREM2 activities comprise an increased        ability of dendritic cells, monocytes, microglia, and/or        macrophages to induce T-cell proliferation.        326. The isolated antibody of any one of embodiments 303-324,        wherein the one or more TREM2 activities comprise an        enhancement, normalization, or both of the ability of bone        marrow-derived dendritic cells to induce antigen-specific T-cell        proliferation.        327. The isolated antibody of any one of embodiments 303-326,        wherein the one or more TREM2 activities comprise induction of        osteoclast production, increased rate of osteoclastogenesis, or        both.        328. The isolated antibody of any one of embodiments 303-327,        wherein the one or more TREM2 activities comprise increasing the        survival of dendritic cells, macrophages, monocytes,        osteoclasts, Langerhans cells of skin, Kupffer cells, and/or        microglia.        329. The isolated antibody of any one of embodiments 303-328,        wherein the one or more TREM2 activities comprise increasing the        function of dendritic cells, macrophages, and/or microglia.        330. The isolated antibody of any one of embodiments 303-329,        wherein the one or more TREM2 activities comprise increasing        phagocytosis by dendritic cells, macrophages, monocytes, and/or        microglia under conditions of reduced levels of MCSF.        331. The isolated antibody of any one of embodiments 303-330,        wherein the one or more TREM2 activities comprise decreasing        phagocytosis by dendritic cells, macrophages, monocytes, and/or        microglia in the presence of normal levels of MCSF.        332. The isolated antibody of any one of embodiments 328-331,        wherein the macrophages and/or microglia are M1 macrophages        and/or microglia, M2 macrophages and/or microglia, or both.        333. The isolated antibody of embodiment 332, wherein the M1        macrophages and/or microglia are activated M1 macrophages and/or        microglia.        334. The isolated antibody of any one of any one of embodiments        303-333, wherein the one or more TREM2 activities comprise        induction of one or more types of clearance selected from the        group consisting of apoptotic neuron clearance, nerve tissue        debris clearance, non-nerve tissue debris clearance, bacteria or        other foreign body clearance, disease-causing protein clearance,        and tumor cell clearance.        335. The isolated antibody of any one of embodiments 303-334,        wherein the one or more TREM2 activities comprise induction of        phagocytosis of one or more of apoptotic neurons, nerve tissue        debris, non-nerve tissue debris, bacteria, other foreign bodies,        disease-causing proteins, disease-causing peptides,        disease-causing nucleic acids, or tumor cells.        336. The isolated antibody of embodiment 335, wherein the        disease-causing nucleic acid is antisense GGCCCC (G2C4)        repeat-expansion RNA.        337. The isolated antibody of embodiment 334 or embodiment 335,        wherein the disease-causing protein is selected from the group        consisting of amyloid beta or fragments thereof, Tau, IAPP,        alpha-synuclein, TDP-43, FUS protein, prion protein, PrPSc,        huntingtin, calcitonin, superoxide dismutase, ataxin, Lewy body,        atrial natriuretic factor, islet amyloid polypeptide, insulin,        apolipoprotein AI, serum amyloid A, medin, prolactin,        transthyretin, lysozyme, beta 2 microglobulin, gelsolin,        keratoepithelin, cystatin, immunoglobulin light chain AL, S-IBM        protein, Repeat-associated non-ATG (RAN) translation products,        DiPeptide repeat (DPR) peptides, glycine-alanine (GA) repeat        peptides, glycine-proline (GP) repeat peptides, glycine-arginine        (GR) repeat peptides, proline-alanine (PA) repeat peptides, and        proline-arginine (PR) repeat peptides.        338. The isolated antibody of any one of embodiments 303-337,        wherein the one or more TREM2 activities comprise normalization        of disrupted TREM2/DAP12-dependent gene expression.        339. The isolated antibody of any one of embodiments 303-338,        wherein the one or more TREM2 activities comprise recruitment of        Syk, ZAP70, or both to a DAP12/TREM2 complex.        340. The isolated antibody of any one of embodiments 303-339,        wherein the one or more TREM2 activities comprise Syk        phosphorylation.        341. The isolated antibody of any one of embodiments 303-340,        wherein the one or more TREM2 activities comprise increased        expression of CD83 and/or CD86 on dendritic cells, monocytes,        macrophages, or both.        342. The isolated antibody of embodiment 341, wherein the        dendritic cells are bone marrow-derived dendritic cells.        343. The isolated antibody of any one of embodiments 303-342,        wherein the one or more TREM2 activities comprise reduced        secretion of one or more inflammatory cytokines.        344. The isolated antibody of 343, wherein the one or more        inflammatory cytokines are selected from the group consisting of        IFN-a4, IFN-b, IL-1β, TNF-α, IL-10, IL-6, IL-8, CRP, TGF-beta        members of the chemokine protein families, IL-20 family members,        IL-33, LIF, IFN-gamma, OSM, CNTF, TGF-beta, GM-CSF, IL-11,        IL-12, IL-17, IL-18, CRP, and MCP-1.        345. The isolated antibody of any one of embodiments 303-344,        wherein the one or more TREM2 activities comprise reduced        expression of one or more inflammatory receptors.        346. The isolated antibody of embodiment 345, wherein the one or        more inflammatory receptors comprise CD86.        347. The isolated antibody of any one of embodiments 303-346,        wherein the one or more TREM2 activities comprise increasing        activity of one or more TREM2-dependent genes.        348. The isolated antibody of embodiment 347, wherein the one or        more TREM2-dependent genes comprise one or more nuclear factor        of activated T-cells (NFAT) transcription factors.        349. The isolated antibody of any one of embodiments 303-348,        wherein the antibody is of the IgG class the IgM class, or the        IgA class.        350. The isolated antibody of embodiment 349, wherein the        antibody is of the IgG class and has an IgG1, IgG2, IgG3, or        IgG4 isotype.        351. The isolated antibody of embodiment 349, wherein the        antibody has an IgG2 isotype.        352. The isolated antibody of embodiment 351, wherein the        antibody comprises a human IgG2 constant region.        353. The isolated antibody of embodiment 352, wherein the human        IgG2 constant region comprises an Fc region.        354. The isolated antibody of any one of embodiments 351-353,        wherein the antibody induces the one or more TREM2 activities        independently of binding to an Fc receptor.        355. The isolated antibody of any one of embodiments 351-353,        wherein the antibody binds an inhibitory Fc receptor.        356. The isolated antibody of embodiment 355, wherein the        inhibitory Fc receptor is inhibitory Fc-gamma receptor IIB        (FcγIIB).        357. The isolated antibody of embodiment 355 or embodiment 356,        wherein the human IgG2 constant region comprises an Fc region        that comprises one or more modifications.        358. The isolated antibody of embodiment 357, wherein the Fc        region comprises one or more amino acid substitutions.        359. The isolated antibody of embodiment 358, wherein the one or        more amino acid substitutions in the Fc region are at a residue        position selected from the group consisting of V234A, G237A,        H268Q, V309L, A330S, P331S, C232S, C233S, S267E, L328F, M252Y,        S254T, T256E, and any combination thereof, wherein the numbering        of the residues is according to EU numbering.        360. The isolated antibody of embodiment 352, wherein the human        IgG2 constant region comprises a light chain constant region        comprising a C214S amino acid substitution, wherein the        numbering of the residues is according to EU numbering.        361. The isolated antibody of embodiment 349, wherein the        antibody has an IgG1 isotype.        362. The isolated antibody of embodiment 361, wherein the        antibody comprises a human IgG1 constant region.        363. The isolated antibody of embodiment 362, wherein the human        IgG1 constant region comprises an Fc region.        364. The isolated antibody of any one of embodiments 361-363,        wherein the antibody binds an inhibitory Fc receptor.        365. The isolated antibody of embodiment 364, wherein the        inhibitory Fc receptor is inhibitory Fc-gamma receptor IIB        (FcγRIIB).        366. The isolated antibody of any one of embodiments 363-365,        wherein the Fc region comprises one or more modifications.        367. The isolated antibody of embodiment 366, wherein the Fc        region comprises one or more amino acid substitutions.        368. The isolated antibody of embodiment 367, wherein the one or        more amino acid substitutions in the Fc region are at a residue        position selected from the group consisting of N297A, D265A,        L234A, L235A, G237A, C226S, C229S, E233P, L234V, L234F, L235E,        P331S, S267E, L328F, A330L, M252Y, S254T, T256E, and any        combination thereof, wherein the numbering of the residues is        according to EU numbering.        369. The isolated antibody of any one of embodiments 363-365,        wherein the antibody comprises an IgG2 isotype heavy chain        constant domain 1(CH1) and hinge region.        370. The isolated antibody of embodiment 369, wherein the IgG2        isotype CH1 and hinge region comprise the amino acid sequence of        ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYHFEPVTVS WNSGALTSGVHTFPAVLQSS        GLYSLSSVVT VPSSNFGTQT YTCNVDHKPS NTKVDKTVERKCCVECPPCP (SEQ ID        NO: 397).        371. The isolated antibody of embodiment 369 or embodiment 370,        wherein the antibody Fc region comprises a S267E amino acid        substitution, a L328F amino acid substitution, or both, and/or a        N297A or N297Q amino acid substitution, wherein the numbering of        the residues is according to EU numbering.        372. The isolated antibody of embodiment 361, wherein the        antibody comprises a mouse IgG1 constant region.        373. The isolated antibody of embodiment 349, wherein the        antibody has an IgG4 isotype.        374. The isolated antibody of embodiment 373, wherein the        antibody comprises a human IgG4 constant region.        375. The isolated antibody of embodiment 374, wherein the human        IgG4 constant region comprises an Fc region.        376. The isolated antibody of any one of embodiments 373-375,        wherein the antibody binds an inhibitory Fc receptor.        377. The isolated antibody of embodiment 376, wherein the        inhibitory Fc receptor is inhibitory Fc-gamma receptor IIB        (FcγIIB).        378. The isolated antibody of any one of embodiments 375-377,        wherein the Fc region comprises one or more modifications.        379. The isolated antibody of embodiment 378, wherein the Fc        region comprises one or more amino acid substitutions.        380. The isolated antibody of embodiment 379, wherein the one or        more amino acid substitutions in the Fc region are at a residue        position selected from the group consisting of L235A, G237A,        S228P, L236E, S267E, E318A, L328F, M252Y, S254T, T256E, and any        combination thereof, wherein the numbering of the residues is        according to EU numbering.        381. The isolated antibody of embodiment 349, wherein the        antibody has a hybrid IgG2/4 isotype.        382. The isolated antibody of embodiment 381, wherein the        antibody comprises an amino acid sequence comprising amino acids        118 to 260 of human IgG2 and amino acids 261 to 447 of human        IgG4, wherein the numbering of the residues is according to EU        numbering.        383. The isolated antibody of embodiment 373, wherein the        antibody comprises a mouse IgG4 constant region.        384. The isolated antibody of any one of embodiments 1-383,        wherein the isolated antibody is an antibody fragment that binds        to one or more human proteins selected from the group consisting        of human TREM2, a naturally occurring variant of human TREM2,        and a disease variant of human TREM2, and wherein the antibody        fragment is cross-linked to a second antibody fragment that        binds to one or more human proteins selected from the group        consisting of human TREM2, a naturally occurring variant of        human TREM2, and a disease variant of human TREM2.        385. The isolated antibody of embodiment 384, wherein the        fragment is an Fab, Fab′, Fab′-SH, F(ab′)2, Fv or scFv fragment.        386. An isolated inert antibody that binds to a TREM2 protein.        387. An isolated antagonist antibody that binds to a TREM2        protein.        388. The isolated antibody of embodiment 386 or embodiment 387,        wherein the TREM2 protein is a mammalian protein or a human        protein.        389. The isolated antibody of embodiment 388, wherein the TREM2        protein is a wild-type protein.        390. The isolated antibody of embodiment 388, wherein the TREM2        protein is a naturally occurring variant.        391. The isolated antibody of embodiment 388, wherein the TREM2        protein is a disease variant.        392. The isolated antibody of any one of embodiments 387-391,        wherein the isolated antibody inhibits one or more TREM2        activities.        393. The isolated antibody of embodiment 392, wherein the one or        more TREM2 activities comprise decreasing activity of one or        more TREM2-dependent genes.        394. The isolated antibody of embodiment 393, wherein the one or        more TREM2-dependent genes comprise one or more nuclear factor        of activated T-cells (NFAT) transcription factors.        395. The isolated antibody of any one of embodiments 392-394,        wherein the one or more TREM2 activities comprise decreasing the        survival of macrophages, microglial cells, M1 macrophages, M1        microglial cells, M2 macrophages, M2 microglial cells,        osteoclasts, Langerhans cells of skin, Kupffer cells, and/or        dendritic cells.        396. The isolated antibody of any one of embodiments 387-395,        wherein the isolated antibody inhibits interaction between TREM2        and one or more TREM2 ligands, inhibits TREM2 signal        transduction, or both.        397. The isolated antibody of any one of embodiments 386-396,        wherein the antibody is incapable of binding an Fc-gamma        receptor (FcγR).        398. The isolated antibody of embodiment 397, wherein the        antibody has an IgG1 isotype.        399. The isolated antibody of embodiment 398, wherein the        antibody comprises a human IgG1 constant region.        400. The isolated antibody of embodiment 399, wherein the human        IgG1 constant region comprises an Fc region.        401. The isolated antibody of embodiment 400, wherein the Fc        region comprises one or more modifications.        402. The isolated antibody of embodiment 401, wherein the Fc        region comprises one or more amino acid substitutions.        403. The isolated antibody of embodiment 402, wherein the one or        more amino acid substitutions in the Fc region are at a residue        position selected from the group consisting of N297A, N297Q,        D265A, L234A, L235A, C226S, C229S, P238S, E233P, L234V, P238A,        A327Q, A327G, P329A, K322A, L234F, L235E, 1P331S, T394D, A330L,        M252Y, S254T, T256E, and any combination thereof, wherein the        numbering of the residues is according to EU numbering.        404. The isolated antibody of embodiment 403, wherein the Fc        region further comprises an amino acid deletion at a position        corresponding to glycine 236 according to EU numbering.        405. The isolated antibody of embodiment 398, wherein the        antibody comprises a mouse IgG1 constant region.        406. The isolated antibody of embodiment 397, wherein the        antibody has an IgG2 isotype.        407. The isolated antibody of embodiment 406, wherein the        antibody comprises a human IgG2 constant region.        408. The isolated antibody of embodiment 407, wherein the human        IgG2 constant region comprises an Fc region.        409. The isolated antibody of embodiment 408, wherein the Fc        region comprises one or more modifications.        410. The isolated antibody of embodiment 409, wherein the Fc        region comprises one or more amino acid substitutions.        411. The isolated antibody of embodiment 410, wherein the one or        more amino acid substitutions in the Fc region are at a residue        position selected from the group consisting of V234A, G237A,        H268E, V309L, N297A, N297Q, A330S, P331S, C232S, C233S, M252Y,        S254T, T256E, and any combination thereof, wherein the numbering        of the residues is according to EU numbering.        412. The isolated antibody of embodiment 397 wherein the        antibody has an IgG4 isotype.        413. The isolated antibody of embodiment 412, wherein the        antibody comprises a human IgG4 constant region.        414. The isolated antibody of embodiment 413, wherein the human        IgG4 constant region comprises an Fc region.        415. The isolated antibody of embodiment 414, wherein the Fc        region comprises one or more modifications.        416. The isolated antibody of embodiment 415, wherein the Fc        region comprises one or more amino acid substitutions.        417. The isolated antibody of embodiment 416, wherein the one or        more amino acid substitutions in the Fc region are at a residue        position selected from the group consisting of E233P, F234V,        L235A, G237A, E318A, S228P, L236E, S241P, L248E, T394D, M252Y,        S254T, T256E, N297A, N297Q, and any combination thereof, wherein        the numbering of the residues is according to EU numbering.        418. The isolated antibody of any one of embodiments 386-417,        wherein the isolated antibody is an antibody fragment that binds        to one or more human proteins selected from the group consisting        of human TREM2, a naturally occurring variant of human TREM2,        and a disease variant of TREM2.        419. The isolated antibody of embodiment 418, wherein the        fragment is an Fab, Fab′, Fab′-SH, F(ab′)2, Fv or scFv fragment.        420. The isolated antibody of embodiment 368, embodiment 403, or        embodiment 404, wherein the Fc region further comprises one or        more additional amino acid substitutions at a position selected        from the group consisting of A330L, L234F; L235E, P331S, and any        combination thereof, wherein the numbering of the residues is        according to EU numbering.        421. The isolated antibody of any one of embodiments 357-420,        wherein the Fc region further comprises one or more additional        amino acid substitutions at a position selected from the group        consisting of M252Y, S254T, T256E, and any combination thereof,        wherein the numbering of the residues is according to EU        numbering.        422. The isolated antibody of embodiment 380 or embodiment 417,        wherein the Fc region further comprises a S228P amino acid        substitution according to EU numbering.        423. The isolated antibody of any one of embodiments 303-422,        wherein the antibody competes for binding of TREM2 with one or        more TREM2 ligands.        424. The isolated antibody of embodiment 423, wherein the one or        more TREM2 ligands are selected from the group consisting of E.        coli cells, apoptotic cells, nucleic acids, anionic lipids,        zwitterionic lipids, negatively charged phospholipids,        phosphatidylserine, sulfatides, phosphatidylcholin,        sphingomyelin, membrane phospholipids, lipidated proteins,        proteolipids, lipidated peptides, and lipidated amyloid beta        peptide.        425. The isolated antibody of any one of embodiments 303-422,        wherein the antibody does not compete for binding of TREM2 with        a TREM2 ligand.        426. The isolated antibody of any one of embodiments 303-425,        wherein the antibody is a human antibody, a humanized antibody,        a bispecific antibody, a multivalent antibody, a conjugated        antibody, or a chimeric antibody.        427. The isolated antibody of any one of embodiments 303-426,        wherein the antibody is a bispecific antibody recognizing a        first antigen and a second antigen.        428. The isolated antibody of embodiment 427, wherein the first        antigen is human TREM2 or a naturally occurring variant thereof,        and the second antigen is a disease-causing protein selected        from the group consisting of: amyloid beta or fragments thereof,        Tau, IAPP, alpha-synuclein, TDP-43, FUS protein, prion protein,        PrPSc, huntingtin, calcitonin, superoxide dismutase, ataxin,        Lewy body, atrial natriuretic factor, islet amyloid polypeptide,        insulin, apolipoprotein AI, serum amyloid A, medin, prolactin,        transthyretin, lysozyme, beta 2 microglobulin, gelsolin,        keratoepithelin, cystatin, immunoglobulin light chain AL, S-IBM        protein, Repeat-associated non-ATG (RAN) translation products,        DiPeptide repeat (DPR) peptides, glycine-alanine (GA) repeat        peptides, glycine-proline (GP) repeat peptides, glycine-arginine        (GR) repeat peptides, proline-alanine (PA) repeat peptides, and        proline-arginine (PR) repeat peptides; a blood brain barrier        targeting protein selected from the group consisting of:        trasnferin receptor, insulin receptor, insulin like growth        factor receptor, LRP-1, and LRP1; or ligands and/or proteins        expressed on immune cells, wherein the ligands and/or proteins        selected from the group consisting of: CD40, OX40, ICOS, CD28,        CD137/4-1BB, CD27, GITR, PD-L1, CTLA4, PD-L2, PD-1, B7-H3,        B7-H4, HVEM, BTLA, KIR, GAL9, TIM3, A2AR, LAG, and        phosphatidylserine.        429. The isolated antibody of embodiment 427, wherein the first        antigen is human TREM2 or a naturally occurring variant thereof,        and the second antigen is a protein expressed on one or more        tumor cells.        430. The isolated antibody of any one of embodiments 303-424,        wherein the antibody is an antibody fragment that binds to one        or more human proteins selected from the group consisting of        human TREM2, a naturally occurring variant of human TREM2, human        DAP12, and naturally occurring variant of human DAP12; and        wherein the antibody is used in combination with one or more        antibodies that specifically bind a disease-causing protein        selected from the group consisting of: amyloid beta, Tau, IAPP,        alpha-synuclein, TDP-43, FUS protein, prion protein, PrPSc,        huntingtin, calcitonin, superoxide dismutase, ataxin, Lewy body,        atrial natriuretic factor, islet amyloid polypeptide, insulin,        apolipoprotein AI, serum amyloid A, medin, prolactin,        transthyretin, lysozyme, beta 2 microglobulin, gelsolin,        keratoepithelin, cystatin, immunoglobulin light chain AL, S-IBM        protein, Repeat-associated non-ATG (RAN) translation products,        DiPeptide repeat (DPR) peptides, glycine-alanine (GA) repeat        peptides, glycine-proline (GP) repeat peptides, glycine-arginine        (GR) repeat peptides, proline-alanine (PA) repeat peptides, and        proline-arginine (PR) repeat peptides, and any combination        thereof, or with one or more antibodies that specifically bind a        cancer-associated protein selected from the group consisting of:        CD40, OX40, ICOS, CD28, CD137/4-1BB, CD27, GITR, PD-L1, CTLA4,        PD-L2, PD-1, B7-H3, B7-H4, HVEM, BTLA, KIR, GAL9, TIM3, A2AR,        LAG, phosphatidylserine, and any combination thereof.        431. The isolated antibody of any one of embodiments 303-430,        wherein the antibody is a monoclonal antibody.        432. The isolated antibody of any one of embodiments 303-431,        wherein the antibody binds to one or more amino acids within        amino acid residues selected from the group consisting of:    -   xi. amino acid residues 130-171 of SEQ ID NO: 1, or amino acid        residues on a TREM2 protein corresponding to amino acid residues        130-171 of SEQ ID NO: 1;    -   xii. amino acid residues 140-153 of SEQ ID NO: 1, or amino acid        residues on a TREM2 protein corresponding to amino acid residues        140-153 of SEQ ID NO: 1;    -   xiii. amino acid residues 139-146 of SEQ ID NO: 1, or amino acid        residues on a TREM2 protein corresponding to amino acid residues        139-146 of SEQ ID NO: 1;    -   xiv. amino acid residues 130-144 of SEQ ID NO: 1, or amino acid        residues on a TREM2 protein corresponding to amino acid residues        130-144 of SEQ ID NO: 1; and    -   xv. amino acid residues 158-171 of SEQ ID NO: 1, or amino acid        residues on a TREM2 protein corresponding to amino acid residues        158-171 of SEQ ID NO: 1.        433. The isolated antibody of any one of embodiments 303-431,        wherein the antibody binds to an epitope comprising one or more        amino acids within amino acid residues selected from the group        consisting of:    -   i. amino acid residues 130-171 of SEQ ID NO: 1, or amino acid        residues on a TREM2 protein corresponding to amino acid residues        130-171 of SEQ ID NO: 1;    -   ii. amino acid residues 140-153 of SEQ ID NO: 1, or amino acid        residues on a TREM2 protein corresponding to amino acid residues        139-153 of SEQ ID NO: 1;    -   iii. amino acid residues 139-146 of SEQ ID NO: 1, or amino acid        residues on a TREM2 protein corresponding to amino acid residues        139-146 of SEQ ID NO: 1;    -   iv. amino acid residues 130-144 of SEQ ID NO: 1, or amino acid        residues on a TREM2 protein corresponding to amino acid residues        130-144 of SEQ ID NO: 1; and    -   v. amino acid residues 158-171 of SEQ ID NO: 1, or amino acid        residues on a TREM2 protein corresponding to amino acid residues        158-171 of SEQ ID NO: 1.        434. The isolated antibody of embodiment 432 or embodiment 433,        wherein the antibody binds to an epitope further comprising one        or more amino acid residues selected from the group consisting        of:    -   v. amino acid residue Arg47 or Asp87 of SEQ ID NO: 1;    -   vi. amino acid residues 40-44 of SEQ ID NO: 1;    -   vii. amino acid residues 67-76 of SEQ ID NO: 1; and    -   viii. amino acid residues 114-118 of SEQ ID NO: 1.        435. The isolated antibody of any one of embodiments 303-433,        wherein the isolated antibody comprises a heavy chain variable        domain and a light chain variable domain,        wherein the heavy chain variable domain comprises an HVR-H1,        HVR-H2, and/or HVR-H3 of a monoclonal antibody selected from the        group consisting of: Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8,        Ab9, Ab10, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19,        Ab20, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30,        Ab31, Ab32, Ab33, Ab34, Ab35, Ab36, Ab37, Ab38, Ab39, Ab40,        Ab41, Ab42, Ab43, Ab44, Ab45, Ab46, Ab47, Ab48, Ab49, Ab50,        Ab51, Ab53, Ab54, Ab55, Ab56, Ab57, Ab58, Ab59, Ab60, Ab61,        Ab62, Ab63, Ab64, Ab65, Ab66, Ab67, Ab68, Ab69, Ab70, Ab71,        Ab72, Ab73, Ab74, Ab75, Ab76, Ab77, Ab78, Ab79, Ab80, Ab81,        Ab82, Ab83, Ab84, Ab85, Ab86, and Ab87, and/or        wherein the light chain variable domain comprises an HVR-L1,        HVR-L2, and/or HVR-L3 of a monoclonal antibody selected from the        group consisting of: Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8,        Ab9, Ab10, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19,        Ab20, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30,        Ab31, Ab32, Ab33, Ab34, Ab35, Ab36, Ab37, Ab38, Ab39, Ab40,        Ab41, Ab42, Ab43, Ab44, Ab45, Ab46, Ab47, Ab48, Ab49, Ab50,        Ab51, Ab53, Ab54, Ab55, Ab56, Ab57, Ab58, Ab59, Ab60, Ab61,        Ab62, Ab63, Ab64, Ab65, Ab66, Ab67, Ab68, Ab69, Ab70, Ab71,        Ab72, Ab73, Ab74, Ab75, Ab76, Ab77, Ab78, Ab79, Ab80, Ab81,        Ab82, Ab83, Ab84, Ab85, Ab86, and Ab87.        436. The isolated antibody of embodiment 435, wherein the HVR-H1        comprises an amino acid sequence selected from the group        consisting of SEQ ID NOs:3-24.        437. The isolated antibody of embodiment 435 or embodiment 436,        wherein the HVR-H2 comprises an amino acid sequence selected        from the group consisting of SEQ ID NOs:25-49.        438. The isolated antibody of any one of embodiments 435-437,        wherein the HVR-H3 comprises an amino acid sequence selected        from the group consisting of SEQ ID NOs:50-119.        439. The isolated antibody of any one of embodiments 435-438,        wherein the HVR-L1 comprises an amino acid sequence selected        from the group consisting of SEQ ID NOs:120-137.        440. The isolated antibody of any one of embodiments 435-439,        wherein the HVR-L2 comprises an amino acid sequence selected        from the group consisting of SEQ ID NOs:138-152.        441. The isolated antibody of any one of embodiments 435-440,        wherein the HVR-L3 comprises an amino acid sequence selected        from the group consisting of SEQ ID NOs:153-236.        442. The isolated antibody of any one of embodiments 303-433,        wherein the isolated antibody comprises a heavy chain variable        domain and a light chain variable domain, wherein the heavy        chain variable domain comprises:    -   (a) an HVR-H1 comprising an amino acid sequence selected from        the group consisting of SEQ ID NOs:3-24, or an amino acid        sequence with at least about 95% homology to an amino acid        sequence selected from the group consisting of SEQ ID NOs:3-24;    -   (b) an HVR-H2 comprising an amino acid sequence selected from        the group consisting of SEQ ID NOs:25-49, or an amino acid        sequence with at least about 95% homology to an amino acid        sequence selected from the group consisting of SEQ ID NOs:        25-49; and    -   (c) an HVR-H3 comprising an amino acid sequence selected from        the group consisting of SEQ ID NOs:50-119, or an amino acid        sequence with at least about 95% homology to an amino acid        sequence selected from the group consisting of SEQ ID NOs:        50-119; and/or        wherein the light chain variable domain comprises:    -   (a) an HVR-L1 comprising an amino acid sequence selected from        the group consisting of SEQ ID NOs:120-137, or an amino acid        sequence with at least about 95% homology to an amino acid        sequence selected from the group consisting of SEQ ID        NOs:120-137;    -   (b) an HVR-L2 comprising an amino acid sequence selected from        the group consisting of SEQ ID NOs:138-152, or an amino acid        sequence with at least about 95% homology to an amino acid        sequence selected from the group consisting of SEQ ID NOs:        138-152; and    -   (c) an HVR-L3 comprising an amino acid sequence selected from        the group consisting of SEQ ID NOs:153-236 or an amino acid        sequence with at least about 95% homology to an amino acid        sequence selected from the group consisting of SEQ ID NOs:        138-152.        443. An isolated anti-human TREM2 antibody, wherein the isolated        antibody comprises a heavy chain variable domain and a light        chain variable domain,        wherein the heavy chain variable domain comprises the HVR-H1,        HVR-H2, and/or HVR-H3 of a monoclonal antibody selected from the        group consisting of: Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8,        Ab9, Ab10, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19,        Ab20, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30,        Ab31, Ab32, Ab33, Ab34, Ab35, Ab36, Ab37, Ab38, Ab39, Ab40,        Ab41, Ab42, Ab43, Ab44, Ab45, Ab46, Ab47, Ab48, Ab49, Ab50,        Ab51, Ab53, Ab54, Ab55, Ab56, Ab57, Ab58, Ab59, Ab60, Ab61,        Ab62, Ab63, Ab64, Ab65, Ab66, Ab67, Ab68, Ab69, Ab70, Ab71,        Ab72, Ab73, Ab74, Ab75, Ab76, Ab77, Ab78, Ab79, Ab80, Ab81,        Ab82, Ab83, Ab84, Ab85, Ab86, and Ab87, and/or        wherein the light chain variable domain comprises the HVR-L1,        HVR-L2, and/or HVR-L3 of a monoclonal antibody selected from the        group consisting of: Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8,        Ab9, Ab10, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19,        Ab20, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30,        Ab31, Ab32, Ab33, Ab34, Ab35, Ab36, Ab37, Ab38, Ab39, Ab40,        Ab41, Ab42, Ab43, Ab44, Ab45, Ab46, Ab47, Ab48, Ab49, Ab50,        Ab51, Ab53, Ab54, Ab55, Ab56, Ab57, Ab58, Ab59, Ab60, Ab61,        Ab62, Ab63, Ab64, Ab65, Ab66, Ab67, Ab68, Ab69, Ab70, Ab71,        Ab72, Ab73, Ab74, Ab75, Ab76, Ab77, Ab78, Ab79, Ab80, Ab81,        Ab82, Ab83, Ab84, Ab85, Ab86, and Ab87.        444. The isolated antibody of embodiment 443, wherein the HVR-H1        comprises an amino acid sequence selected from the group        consisting of SEQ ID NOs:3-24.        445. The isolated antibody of embodiment 443 or embodiment 444,        wherein the HVR-H2 comprises an amino acid sequence selected        from the group consisting of SEQ ID NOs:25-49.        446. The isolated antibody of any one of embodiments 443-445,        wherein the HVR-H3 comprises an amino acid sequence selected        from the group consisting of SEQ ID NOs:50-119.        447. The isolated antibody of any one of embodiments 443-446,        wherein the HVR-L1 comprises an amino acid sequence selected        from the group consisting of SEQ ID NOs:120-137.        448. The isolated antibody of any one of embodiments 443-447,        wherein the HVR-L2 comprises an amino acid sequence selected        from the group consisting of SEQ ID NOs:138-152.        449. The isolated antibody of any one of embodiments 443-448,        wherein the HVR-L3 comprises an amino acid sequence selected        from the group consisting of SEQ ID NOs:153-236.        450. The isolated antibody of embodiment 443, wherein the        isolated antibody comprises a heavy chain variable domain and a        light chain variable domain, wherein the heavy chain variable        domain comprises an HVR-H1 comprising an amino acid sequence        selected from the group consisting of SEQ ID NOs:3-24, an HVR-H2        comprising an amino acid sequence selected from the group        consisting of SEQ ID NOs:25-49, and an HVR-H3 comprising an        amino acid sequence selected from the group consisting of SEQ ID        NOs:50-119, and/or wherein the light chain variable domain        comprises an HVR-L1 comprising an amino acid sequence selected        from the group consisting of SEQ ID NOs:120-137, an HVR-L2        comprising an amino acid sequence selected from the group        consisting of SEQ ID NOs:138-152, and an HVR-L3 comprising an        amino acid sequence selected from the group consisting of SEQ ID        NOs:153-236.        451. An isolated anti-human TREM2 antibody which binds        essentially the same TREM2 epitope as a monoclonal antibody        selected from the group consisting of: Ab1, Ab2, Ab3, Ab4, Ab5,        Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16,        Ab17, Ab18, Ab19, Ab20, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27,        Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, Ab34, Ab35, Ab36, Ab37,        Ab38, Ab39, Ab40, Ab41, Ab42, Ab43, Ab44, Ab45, Ab46, Ab47,        Ab48, Ab49, Ab50, Ab51, Ab53, Ab54, Ab55, Ab56, Ab57, Ab58,        Ab59, Ab60, Ab61, Ab62, Ab63, Ab64, Ab65, Ab66, Ab67, Ab68,        Ab69, Ab70, Ab71, Ab72, Ab73, Ab74, Ab75, Ab76, Ab77, Ab78,        Ab79, Ab80, Ab81, Ab82, Ab83, Ab84, Ab85, Ab86, and Ab87.        452. An isolated anti-human TREM2 antibody, wherein the isolated        antibody comprises a heavy chain variable domain and a light        chain variable domain, wherein the heavy chain variable domain        comprises:    -   (a) an HVR-H1 comprising an amino acid sequence selected from        the group consisting of SEQ ID NOs:3-24, or an amino acid        sequence with at least about 95% homology to an amino acid        sequence selected from the group consisting of SEQ ID NOs:3-24;    -   (b) an HVR-H2 comprising an amino acid sequence selected from        the group consisting of SEQ ID NOs:25-49, or an amino acid        sequence with at least about 95% homology to an amino acid        sequence selected from the group consisting of SEQ ID NOs:        25-49; and    -   (c) an HVR-H3 comprising an amino acid sequence selected from        the group consisting of SEQ ID NOs:50-119, or an amino acid        sequence with at least about 95% homology to an amino acid        sequence selected from the group consisting of SEQ ID NOs:        50-119; and/or        wherein the light chain variable domain comprises:    -   (a) an HVR-L1 comprising an amino acid sequence selected from        the group consisting of SEQ ID NOs:120-137, or an amino acid        sequence with at least about 95% homology to an amino acid        sequence selected from the group consisting of SEQ ID        NOs:120-137;    -   (b) an HVR-L2 comprising an amino acid sequence selected from        the group consisting of SEQ ID NOs:138-152, or an amino acid        sequence with at least about 95% homology to an amino acid        sequence selected from the group consisting of SEQ ID NOs:        138-152; and    -   (c) an HVR-L3 comprising an amino acid sequence selected from        the group consisting of SEQ ID NOs:153-236 or an amino acid        sequence with at least about 95% homology to an amino acid        sequence selected from the group consisting of SEQ ID NOs:        138-152.        453. The isolated antibody of any one of embodiments 443-452,        wherein the antibody is an agonist antibody, and wherein the        antibody induces one or more TREM2 activities.        454. The isolated antibody of embodiment 453, wherein the        isolated antibody induces or retains TREM2 clustering on a cell        surface.        455. The isolated antibody of embodiment 453 or embodiment 454,        wherein the one or more TREM2 activities are selected from the        group consisting of TREM2 binding to DAP12; DAP12 binding to        TREM2; TREM2 phosphorylation, DAP12 phosphorylation; PI3K        activation; increased expression of one or more cytokines        selected from the group consisting of IL-12p70, IL-6, and IL-10;        reduced expression of one or more pro-inflammatory mediators        selected from the group consisting of IFN-a4, IFN-b, IL-1β,        TNF-α, IL-10, IL-6, IL-8, CRP, TGF-beta members of the chemokine        protein families, IL-20 family members, IL-33, LIF, IFN-gamma,        OSM, CNTF, TGF-beta, GM-CSF, IL-11, IL-12, IL-17, IL-18, and        CRP; reduced expression of TNF-α, IL-6, or both; extracellular        signal-regulated kinase (ERK) phosphorylation; increased        expression of C-C chemokine receptor 7 (CCR7); induction of        microglial cell chemotaxis toward CCL19 and CCL21 expressing        cells; increased ability of dendritic cells, monocytes,        microglia, and/or macrophages to induce T-cell proliferation; an        increase, normalization, or both of the ability of bone        marrow-derived dendritic cells to induce antigen-specific T-cell        proliferation; induction of osteoclast production, increased        rate of osteoclastogenesis, or both; increasing the survival        and/or function of one or more of dendritic cells, macrophages,        M1 macrophages, activated M1 macrophages M2 macrophages,        osteoclasts, Langerhans cells of skin, Kupffer cells, microglial        cells, M1 microglial cells, activated M1 microglial cells, and        M2 microglial cells; induction of one or more types of clearance        selected from the group consisting of cancer cells clearance,        apoptotic neuron clearance, nerve tissue debris clearance,        non-nerve tissue debris clearance, bacteria or other foreign        body clearance, and disease-causing protein clearance; induction        of phagocytosis of one or more of apoptotic neurons, nerve        tissue debris, non-nerve tissue debris, bacteria, other foreign        bodies, disease-causing proteins or tumor cells; normalization        of disrupted TREM2/DAP12-dependent gene expression; recruitment        of Syk, ZAP70, or both to the TREM2/DAP12 complex; Syk        phosphorylation; increased expression of CD83 and/or CD86 on        dendritic cells, microglia, monocytes, or macrophages; reduced        secretion of one or more inflammatory cytokines selected from        the group consisting of IFN-a4, IFN-b, IL-1β, TNF-α, IL-10,        IL-6, IL-8, CRP, TGF-beta members of the chemokine protein        families, IL-20 family members, IL-33, LIF, IFN-gamma, OSM,        CNTF, TGF-beta, GM-CSF, IL-11, IL-12, IL-17, IL-18, CRP, and        MCP-1; reduced expression of one or more inflammatory receptors;        increasing phagocytosis by macrophages, monocytes, dendritic        cells, and/or microglia under conditions of reduced levels of        MCSF; decreasing phagocytosis by macrophages, monocytes,        dendritic cells, and/or microglia in the presence of normal        levels of MCSF; increasing activity of one or more        TREM2-dependent genes; and any combination thereof.        456. The isolated antibody of any one of embodiments 453-455,        wherein the antibody is of the IgG class the IgM class, or the        IgA class.        457. The isolated antibody of embodiment 456, wherein the        antibody is of the IgG class and has an IgG1, IgG2, IgG3, or        IgG4 isotype.        458. The isolated antibody of embodiment 457, wherein the        antibody has an IgG2 isotype.        459. The isolated antibody of embodiment 458, wherein the        antibody comprises a human IgG2 constant region.        460. The isolated antibody of embodiment 459, wherein the human        IgG2 constant region comprises an Fc region.        461. The isolated antibody of any one of embodiments 458-460,        wherein the antibody induces the one or more TREM2 activities        independently of binding to an Fc receptor.        462. The isolated antibody of any one of embodiments 458-461,        wherein the antibody binds an inhibitory Fc receptor.        463. The isolated antibody of embodiment 462, wherein the        inhibitory Fc receptor is inhibitory Fc-gamma receptor IIB        (FcγIIB).        464. The isolated antibody of embodiment 462 or embodiment 463,        wherein the Fc region comprises one or more modifications.        465. The isolated antibody of embodiment 464, wherein the Fc        region comprises one or more amino acid substitutions.        466. The isolated antibody of embodiment 465, wherein the one or        more amino acid substitutions in the Fc region are at a residue        position selected from the group consisting of V234A, G237A,        H268Q, V309L, A330S, P331S, C232S, C233S, S267E, L328F, M252Y,        S254T, T256E, and any combination thereof, wherein the numbering        of the residues is according to EU numbering.        467. The isolated antibody of embodiment 459, wherein the human        IgG2 constant region comprises a light chain constant region        comprising a C214S amino acid substitution, wherein the        numbering of the residues is according to EU numbering.        468. The isolated antibody of embodiment 457, wherein the        antibody has an IgG1 isotype.        469. The isolated antibody of embodiment 468, wherein the        antibody comprises a human IgG1 constant region.        470. The isolated antibody of embodiment 469, wherein the human        IgG1 constant region comprises an Fc region.        471. The isolated antibody of any one of embodiments 468-470,        wherein the antibody binds an inhibitory Fc receptor.        472. The isolated antibody of embodiment 471, wherein the        inhibitory Fc receptor is inhibitory Fc-gamma receptor IIB        (FcγRIIB).        473. The isolated antibody of any one of embodiments 470-472,        wherein the Fc region comprises one or more modifications.        474. The isolated antibody of embodiment 473, wherein the Fc        region comprises one or more amino acid substitutions.        475. The isolated antibody of embodiment 474, wherein the one or        more amino acid substitutions in the Fc region are at a residue        position selected from the group consisting of N297A, D265A,        L234A, L235A, G237A, C226S, C229S, E233P, L234V, L234F, L235E,        P331 S, S267E, L328F, A330L, M252Y, S254T, T256E, and any        combination thereof, wherein the numbering of the residues is        according to EU numbering.        476. The isolated antibody of any one of embodiments 470-472,        wherein the antibody comprises an IgG2 isotype heavy chain        constant domain 1(CH1) and hinge region.        477. The isolated antibody of embodiment 476, wherein the IgG2        isotype CH1 and hinge region comprise the amino acid sequence of        ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGVHTFPAVLQSS        GLYSLSSVVT VPSSNFGTQT YTCNVDHKPS NTKVDKTVERKCCVECPPCP (SEQ ID        NO:397).        478. The isolated antibody of embodiment 476 or embodiment 477,        wherein the antibody Fc region comprises a S267E amino acid        substitution, a L328F amino acid substitution, or both, and/or a        N297A or N297Q amino acid substitution, wherein the numbering of        the residues is according to EU numbering.        479. The isolated antibody of embodiment 468, wherein the        antibody comprises a mouse IgG1 constant region.        480. The isolated antibody of embodiment 457, wherein the        antibody has an IgG4 isotype.        481. The isolated antibody of embodiment 480, wherein the        antibody comprises a human IgG4 constant region.        482. The isolated antibody of embodiment 481, wherein the human        IgG4 constant region comprises an Fc region.        483. The isolated antibody of any one of embodiments 480-482,        wherein the antibody binds an inhibitory Fc receptor.        484. The isolated antibody of embodiment 483, wherein the        inhibitory Fc receptor is inhibitory Fc-gamma receptor IIB        (FcγIIB).        485. The isolated antibody of any one of embodiments 482-484,        wherein the Fc region comprises one or more modifications.        486. The isolated antibody of embodiment 485, wherein the Fc        region comprises one or more amino acid substitutions.        487. The isolated antibody of embodiment 486, wherein the one or        more amino acid substitutions in the Fc region are at a residue        position selected from the group consisting of L235A, G237A,        S228P, L236E, S267E, E318A, L328F, M252Y, S254T, T256E, and any        combination thereof, wherein the numbering of the residues is        according to EU numbering.        488. The isolated antibody of embodiment 456, wherein the        antibody has a hybrid IgG2/4 isotype.        489. The isolated antibody of embodiment 488, wherein the        antibody comprises an amino acid sequence comprising amino acids        118 to 260 of human IgG2 and amino acids 261 to 447 of human        IgG4, wherein the numbering of the residues is according to EU        numbering.        490. The isolated antibody of embodiment 481, wherein the        antibody comprises a mouse IgG4 constant region.        491. The isolated antibody of any one of embodiments 451-490,        wherein the isolated antibody is an antibody fragment that binds        to one or more human proteins selected from the group consisting        of human TREM2, a naturally occurring variant of human TREM2,        and a disease variant of TREM2, and wherein the antibody        fragment is cross-linked to a second antibody fragment that        binds to one or more human proteins selected from the group        consisting of human TREM2, a naturally occurring variant of        human TREM2, and a disease variant of TREM2.        492. The isolated antibody of embodiment 491, wherein the        fragment is an Fab, Fab′, Fab′-SH, F(ab′)2, Fv or scFv fragment.        493. The isolated antibody of any one of embodiments 443-452,        wherein the isolated antibody is an inert antibody.        494. The isolated antibody of any one of embodiments 443-452,        wherein the isolated antibody is an antagonist antibody.        495. The isolated antibody of embodiment 494, wherein the        isolated antibody inhibits one or more TREM2 activities.        496. The isolated antibody of embodiment 495, wherein the one or        more TREM2 activities are selected from the group consisting of        decreasing activity of one or more TREM2-dependent genes;        decreasing activity of one or more nuclear factor of activated        T-cells (NFAT) transcription factors; decreasing the survival of        macrophages, microglial cells, monocytes, osteoclasts,        Langerhans cells of skin, Kupffer cells, and/or dendritic cells;        and any combination thereof.        497. The isolated antibody of any one of embodiments 494-496,        wherein the isolated antibody inhibits interaction between TREM2        and one or more TREM2 ligands, inhibits TREM2 signal        transduction, or both.        498. The isolated antibody of any one of embodiments 493-497,        wherein the antibody is incapable of binding an Fc-gamma        receptor (FcγR).        499. The isolated antibody of embodiment 498, wherein the        antibody has an IgG1 isotype.        500. The isolated antibody of embodiment 499, wherein the        antibody comprises a human IgG1 constant region.        501. The isolated antibody of embodiment 500, wherein the human        IgG1 constant region comprises an Fc region.        502. The isolated antibody of embodiment 501, wherein the Fc        region comprises one or more modifications.        503. The isolated antibody of embodiment 502, wherein the Fc        region comprises one or more amino acid substitutions.        504. The isolated antibody of embodiment 503, wherein the one or        more amino acid substitutions in the Fc region are at a residue        position selected from the group consisting of N297A, N297Q,        D265A, L234A, L235A, C226S, C229S, P238S, E233P, L234V, P238A,        A327Q, A327G, P329A, K322A, L234F, L235E, P331S, T394D, A330L,        M252Y, S254T, T256E, and any combination thereof, wherein the        numbering of the residues is according to EU numbering.        505. The isolated antibody of embodiment 504, wherein the Fc        region further comprises an amino acid deletion at a position        corresponding to glycine 236 according to EU numbering.        506. The isolated antibody of embodiment 499, wherein the        antibody comprises a mouse IgG1 constant region.        507. The isolated antibody of embodiment 498, wherein the        antibody has an IgG2 isotype.        508. The isolated antibody of embodiment 507, wherein the        antibody comprises a human IgG2 constant region.        509. The isolated antibody of embodiment 508, wherein the human        IgG2 constant region comprises an Fc region.        510. The isolated antibody of embodiment 509, wherein the Fc        region comprises one or more modifications.        511. The isolated antibody of embodiment 510, wherein the Fc        region comprises one or more amino acid substitutions.        512. The isolated antibody of embodiment 511, wherein the one or        more amino acid substitutions in the Fc region are at a residue        position selected from the group consisting of V234A, G237A,        H268E, V309L, N297A, N297Q, A330S, P331S, C232S, C233S, M252Y,        S254T, T256E, and any combination thereof, wherein the numbering        of the residues is according to EU numbering.        513. The isolated antibody of embodiment 498, wherein the        antibody has an IgG4 isotype.        514. The isolated antibody of embodiment 513, wherein the        antibody comprises a human IgG4 constant region.        515. The isolated antibody of embodiment 514, wherein the human        IgG4 constant region comprises an Fc region.        516. The isolated antibody of embodiment 515, wherein the Fc        region comprises one or more modifications.        517. The isolated antibody of embodiment 516, wherein the Fe        region comprises one or more amino acid substitutions.        518. The isolated antibody of embodiment 517, wherein the one or        more amino acid substitutions in the Fc region are at a residue        position selected from the group consisting of E233P, F234V,        L235A, G237A, E318A, S228P, L236E, S241P, L248E, T394D, M252Y,        S254T, T256E, N297A, N297Q, and any combination thereof, wherein        the numbering of the residues is according to EU numbering.        519. The isolated antibody of any one of embodiments 443-518,        wherein the isolated antibody is an antibody fragment that binds        to one or more human proteins selected from the group consisting        of human TREM2, a naturally occurring variant of human TREM2,        and a disease variant of TREM2.        520. The isolated antibody of embodiment 519, wherein the        fragment is an Fab, Fab′, Fab′-SH, F(ab′)2, Fv or scFv fragment.        521. The isolated antibody of embodiment 475, embodiment 504, or        embodiment 505, wherein the Fc region further comprises one or        more additional amino acid substitutions at a position selected        from the group consisting of A330L, L234F; L235E, P331S, and any        combination thereof, wherein the numbering of the residues is        according to EU numbering.        522. The isolated antibody of any one of embodiments 465-521,        wherein the Fc region further comprises one or more additional        amino acid substitutions at a position selected from the group        consisting of M252Y, S254T, T256E, and any combination thereof,        wherein the numbering of the residues is according to EU        numbering.        523. The isolated antibody of embodiment 487 or embodiment 518,        wherein the Fc region further comprises a serine to proline        amino acid substitution at position 228 according to EU        numbering.        524. The isolated antibody of any one of embodiments 443-523,        wherein the antibody is a human antibody, a humanized antibody,        a bispecific antibody, a multivalent antibody, or a chimeric        antibody.        525. The isolated antibody of any one of embodiments 443-524,        wherein the antibody is a bispecific antibody recognizing a        first antigen and a second antigen.        526. The isolated antibody of any one of embodiments 443-525,        wherein the antibody is a monoclonal antibody.        527. The isolated antibody of any one of the preceding        embodiments, wherein the isolated antibody binds specifically to        both human TREM2 and mouse TREM2.        528. The isolated antibody of any one of the preceding        embodiments, wherein the isolated antibody has dissociation        constant (K_(D)) for human TREM2 and mouse TREM2 that ranges        from less than about 6.70 nM to less than about 0.23 nM.        529. The isolated antibody of any one of the preceding        embodiments, wherein the isolated antibody has dissociation        constant (K_(D)) for human TREM2-Fc fusion protein that ranges        from less than about 0.71 nM to less than about 0.23 nM.        530. The isolated antibody of any one of the preceding        embodiments, wherein the isolated antibody has dissociation        constant (K_(D)) for human monomeric TREM2 protein that ranges        from less than about 6.70 nM to less than about 0.66 nM.        531. The isolated antibody of any one of the preceding        embodiments, wherein the isolated antibody has dissociation        constant (K_(D)) for mouse TREM2-Fc fusion protein that ranges        from less than about 4.90 nM to less than about 0.35 nM.        532. An isolated nucleic acid encoding the antibody of any one        of the preceding embodiments.        533. A vector comprising the nucleic acid of embodiment 532.        534. A host cell comprising the vector of embodiment 533.        535. A method of producing an antibody, comprising culturing the        cell of embodiment 534 so that the antibody is produced.        536. The method of embodiment 535, further comprising recovering        the antibody produced by the cell.        537. A pharmaceutical composition comprising the antibody of any        one of embodiments 303-531 and a pharmaceutically acceptable        carrier.        538. A method of preventing, reducing risk, or treating an        individual having a disease, disorder, or injury selected from        the group consisting of dementia, frontotemporal dementia,        Alzheimer's disease, vascular dementia, mixed dementia,        Creutzfeldt-Jakob disease, normal pressure hydrocephalus,        amyotrophic lateral sclerosis, Huntington's disease, Taupathy        disease, Nasu-Hakola disease, stroke, acute trauma, chronic        trauma, lupus, acute and chronic colitis, wound healing, Crohn's        disease, inflammatory bowel disease, ulcerative colitis,        obesity, Malaria, essential tremor, central nervous system        lupus, Behcet's disease, Parkinson's disease, dementia with Lewy        bodies, multiple system atrophy, Shy-Drager syndrome,        progressive supranuclear palsy, cortical basal ganglionic        degeneration, acute disseminated encephalomyelitis,        granulomartous disorders, Sarcoidosis, diseases of aging,        seizures, spinal cord injury, traumatic brain injury, age        related macular degeneration, glaucoma, retinitis pigmentosa,        retinal degeneration, respiratory tract infection, sepsis, eye        infection, systemic infection, lupus, arthritis, multiple        sclerosis, low bone density, osteoporosis, osteogenesis,        osteopetrotic disease, Paget's disease of bone, and cancer,        comprising administering to the individual a therapeutically        effective amount of an isolated antibody that binds to a TREM2        protein.        539. A method of inducing or promoting innate immune cell        survival or wound healing an individual in need thereof,        comprising administering to the individual a therapeutically        effective amount of an isolated agonist antibody that binds to a        TREM2 protein.        540. The method of embodiment 538 or embodiment 539, wherein the        isolated antibody is:    -   i. (a) an agonist antibody;        -   (b) an inert antibody; or an        -   (c) an antagonist antibody.            541. The method of embodiment 540, wherein:    -   i. (a) the antibody is of the IgG class the IgM class, or the        IgA class; and/or    -   ii. (b) the antibody has an IgG1, IgG2, IgG3, or IgG4 isotype        542. The method of embodiment 541, wherein the antibody        comprises one or more amino acid substitutions in the Fc region        are at a residue position selected from the group consisting of:    -   i. (a) V234A, G237A, H268Q, V309L, A330S, P331S, C232S, C233S,        S267E, L328F, M252Y, S254T, T256E, and any combination thereof;    -   ii. (b) N297A, D265A, L234A, L235A, G237A, C226S, C229S, E233P,        L234V, L234F, L235E, P331S, S267E, L328F, A330L, M252Y, S254T,        T256E, and any combination thereof;    -   iii. (c) L235A, G237A, S228P, L236E, S267E, E318A, L328F, M252Y,        S254T, T256E, and any combination thereof;    -   iv. (d) N297A, N297Q, D265A, L234A, L235A, C226S, C229S, P238S,        E233P, L234V, P238A, A327Q, A327G, P329A, K322A, L234F, L235E,        P331S, T394D, A330L, M252Y, S254T, T256E, and any combination        thereof;    -   v. (e) V234A, G237A, H268E, V309L, N297A, N297Q, A330S, P331S,        C232S, C233S, M252Y, S254T, T256E, and any combination thereof;        or vi. (f) E233P, F234V, L235A, G237A, E318A, S228P, L236E,        S241P, L248E, T394D, M252Y, S254T, T256E, N297A, N297Q, and any        combination thereof,    -   vii. wherein the numbering of the residues is according to EU        numbering.        543. The method of ant one of embodiments 538-542, wherein the        isolated antibody:    -   i. (a) binds to one or more amino acids within amino acid        residues 43-50 of SEQ ID NO: 1, or amino acid residues on a        TREM2 protein corresponding to amino acid residues 43-50 of SEQ        ID NO: 1; or    -   ii. (b) one or more amino acids within amino acid residues 49-57        of SEQ ID NO: 1, or amino acid residues on a TREM2 protein        corresponding to amino acid residues 49-57 of SEQ ID NO: 1.        544. The method of ant one of embodiments 538-543, wherein the        isolated antibody:    -   i. (a) binds essentially the same TREM2 epitope as the antibody        Ab52;    -   ii. (b) comprises a heavy chain variable domain and a light        chain variable domain,        wherein the heavy chain variable domain comprises the HVR-H1,        HVR-H2, and/or HVR-H3 of the monoclonal antibody Ab52; and/or        wherein the light chain variable domain comprises the HVR-L1,        HVR-L2, and/or HVR-L3 of the monoclonal antibody Ab52;    -   iii. (c) comprises a heavy chain variable domain and a light        chain variable domain,        wherein the heavy chain variable domain comprises an HVR-H1        comprising the amino acid sequence of SEQ ID NO:398, or an amino        acid sequence with at least about 95% homology to the amino acid        sequence of SEQ ID NO:398, an HVR-H2 comprising the amino acid        sequence of SEQ ID NO:399 or an amino acid sequence with at        least about 95% homology to the amino acid sequence of SEQ ID        NO:399, and an HVR-H3 comprising the amino acid sequence of SEQ        ID NO:400 or an amino acid sequence with at least about 95%        homology to the amino acid sequence of SEQ ID NO:400, and/or        wherein the light chain variable domain comprises an HVR-L1        comprising the amino acid sequence of SEQ ID NO:401 or an amino        acid sequence with at least about 95% homology to the amino acid        sequence of SEQ ID NO:401, an HVR-L2 comprising the amino acid        sequence of SEQ ID NO:402 or an amino acid sequence with at        least about 95% homology to the amino acid sequence of SEQ ID        NO:402, and an HVR-L3 comprising the amino acid sequence of SEQ        ID NO:403 or an amino acid sequence with at least about 95%        homology to the amino acid sequence of SEQ ID NO:403;    -   iv. (d) binds essentially the same TREM2 epitope as the antibody        Ab21;    -   v. (e) comprises a heavy chain variable domain and a light chain        variable domain,        wherein the heavy chain variable domain comprises the HVR-H1,        HVR-H2, and/or HVR-H3 of the monoclonal antibody Ab21; and/or        wherein the light chain variable domain comprises the HVR-L1,        HVR-L2, and/or HVR-L3 of the monoclonal antibody Ab21; or    -   vi. (f) comprises a heavy chain variable domain and a light        chain variable domain,        wherein the heavy chain variable domain comprises an HVR-H1        comprising the amino acid sequence of SEQ ID NO:404 or an amino        acid sequence with at least about 95% homology to the amino acid        sequence of SEQ ID NO:404, an HVR-H2 comprising the amino acid        sequence of SEQ ID NO:405 or an amino acid sequence with at        least about 95% homology to the amino acid sequence of SEQ ID        NO:405, and an HVR-H3 comprising the amino acid sequence of SEQ        ID NO:406 or an amino acid sequence with at least about 95%        homology to the amino acid sequence of SEQ ID NO:406, and/or        wherein the light chain variable domain comprises an HVR-L1        comprising the amino acid sequence of SEQ ID NO:407 or an amino        acid sequence with at least about 95% homology to the amino acid        sequence of SEQ ID NO:407, an HVR-L2 comprising the amino acid        sequence of SEQ ID NO:408 or an amino acid sequence with at        least about 95% homology to the amino acid sequence of SEQ ID        NO:408, and an HVR-L3 comprising the amino acid sequence of SEQ        ID NO:409 or an amino acid sequence with at least about 95%        homology to the amino acid sequence of SEQ ID NO:409.        545. The method of embodiment 538, wherein the isolated antibody        is the isolated antibody of any one of embodiments 303-531.        546. The method of embodiment 539, wherein the isolated agonist        antibody is the isolated antibody of any one of embodiments        303-385, 420-492, and 519-531.        547. The method of any one of embodiments 538 and 540-545,        wherein the individual has a heterozygous variant of TREM2,        wherein the variant comprises one or more substitutions selected        from the group consisting of:    -   viii. a glutamic acid to stop codon substitution in the nucleic        acid sequence encoding amino acid residue Glu14 of SEQ ID NO: 1;    -   ix. a glutamine to stop codon substitution in the nucleic acid        sequence encoding amino acid residue Gln33 of SEQ ID NO: 1;    -   x. a tryptophan to stop codon substitution in the nucleic acid        sequence encoding amino acid residue Trp44 of SEQ ID NO: 1;    -   xi. an arginine to histidine amino acid substitution at an amino        acid corresponding to amino acid residue Arg47 of SEQ ID NO: 1;    -   xii. a tryptophan to stop codon substitution in the nucleic acid        sequence encoding amino acid residue Trp78 of SEQ ID NO: 1;    -   xiii. a valine to glycine amino acid substitution at an amino        acid corresponding to amino acid residue Val126 of SEQ ID NO: 1;    -   xiv. an aspartic acid to glycine amino acid substitution at an        amino acid corresponding to amino acid residue Asp134 of SEQ ID        NO: 1; and    -   viii. a lysine to asparagine amino acid substitution at an amino        acid corresponding to amino acid residue Lys186 of SEQ ID NO: 1.        548. The method of any one of embodiments 538, 540-545, and 547,        wherein the individual has a heterozygous variant of TREM2,        wherein the variant comprises a guanine nucleotide deletion at a        nucleotide corresponding to nucleotide residue G313 of the        nucleic acid sequence encoding SEQ ID NO: 1; a guanine        nucleotide deletion at a nucleotide corresponding to nucleotide        residue G267 of the nucleic acid sequence encoding SEQ ID NO: 1;        or both.        549. The method of any one of embodiments 538, 540-545, and        547-548, wherein the individual has a heterozygous variant of        DAP12, wherein the variant comprises one or more variants        selected from the group consisting of:    -   vi. a methionine to threonine substitution at an amino acid        corresponding to amino acid residue Met1 of SEQ ID NO: 2;    -   vii. a glycine to arginine amino acid substitution at an amino        acid corresponding to amino acid residue Gly49 of SEQ ID NO: 2;    -   viii. a deletion within exons 1-4 of the nucleic acid sequence        encoding SEQ ID NO: 2;    -   ix. an insertion of 14 amino acid residues at exon 3 of the        nucleic acid sequence encoding SEQ ID NO: 2; and    -   x. a guanine nucleotide deletion at a nucleotide corresponding        to nucleotide residue G141 of the nucleic acid sequence encoding        SEQ ID NO: 2.        550. The method of any one of embodiments 538 and 540-545,        wherein the cancer is selected from the group consisting of        bladder cancer, brain cancer, breast cancer, colon cancer,        rectal cancer, endometrial cancer, kidney cancer, renal cell        cancer, renal pelvis cancer, leukemia, lung cancer, melanoma,        non-Hodgkin's lymphoma, pancreatic cancer, prostate cancer,        ovarian cancer, fibrosarcoma, and thyroid cancer.        551. The method of any one of embodiments 538 and 540-545, and        550, further comprising administering to the individual at least        one antibody that specifically binds to an inhibitory checkpoint        molecule, and/or another standard or investigational anti-cancer        therapy.        552. The method of embodiment 551, wherein the at least one        antibody that specifically binds to an inhibitory checkpoint        molecule is administered in combination with the isolated        antibody.        553. The method of embodiment 551 or embodiment 552, wherein the        at least one antibody that specifically binds to an inhibitory        checkpoint molecule is selected from the group consisting of an        anti-PD-L1 antibody, an anti-CTLA4 antibody, an anti-PD-L2        antibody, an anti-PD-1 antibody, an anti-B7-H3 antibody, an        anti-B7-H4 antibody, and anti-HVEM antibody, an anti-B- and        T-lymphocyte attenuator (BTLA) antibody, an anti-Killer        inhibitory receptor (KIR) antibody, an anti-GAL9 antibody, an        anti-TIM3 antibody, an anti-A2AR antibody, an anti-LAG-3        antibody, an anti-phosphatidylserine antibody, an anti-CD27        antibody, and any combination thereof.        554. The method of embodiment 551, wherein the standard or        investigational anti-cancer therapy is one or more therapies        selected from the group consisting of radiotherapy, cytotoxic        chemotherapy, targeted therapy, imatinib (Gleevec®), trastuzumab        (Herceptin®), adoptive cell transfer (ACT), chimeric antigen        receptor T cell transfer (CAR-T), vaccine therapy, and cytokine        therapy.        555. The method of any one of embodiments 538, 540-545, and 550,        further comprising administering to the individual at least one        antibody that specifically binds to an inhibitory cytokine.        556. The method of embodiment 555, wherein the at least one        antibody that specifically binds to an inhibitory cytokine is        administered in combination with the isolated antibody.        557. The method of embodiment 555 or embodiment 556, wherein the        at least one antibody that specifically binds to an inhibitory        cytokine is selected from the group consisting of an anti-CCL2        antibody, an anti-CSF-1 antibody, an anti-IL-2 antibody, and any        combination thereof.        558. The method of any one of embodiments 538, 540-545, and 550,        further comprising administering to the individual at least one        agonistic antibody that specifically binds to a stimulatory        checkpoint protein.        559. The method of embodiment 558, wherein the at least one        agonistic antibody that specifically binds to a stimulatory        checkpoint protein is administered in combination with the        isolated antibody.        560. The method of embodiment 558 or embodiment 559, wherein the        at least one agonistic antibody that specifically binds to a        stimulatory checkpoint protein is selected from the group        consisting of an agonist anti-CD40 antibody, an agonist        anti-OX40 antibody, an agonist anti-ICOS antibody, an agonist        anti-CD28 antibody, an agonist anti-CD137/4-1BB antibody, an        agonist anti-CD27 antibody, an agonist        anti-glucocorticoid-induced TNFR-related protein GITR antibody,        and any combination thereof.        561. The method of any one of embodiments 538, 540-545, and 550,        further comprising administering to the individual at least one        stimulatory cytokine.        562. The method of embodiment 561, wherein the at least one        stimulatory cytokine is administered in combination with the        isolated antibody.        563. The method of embodiment 561 or embodiment 562, wherein the        at least one stimulatory cytokine is selected from the group        consisting of TNF-α, IL-10, IL-6, IL-8, CRP, TGF-beta members of        the chemokine protein families, IL20 family member, IL-33, LIF,        OSM, CNTF, TGF-beta, IL-11, IL-12, IL-17, IL-8, CRP, IFN-α,        IFN-β, IL-2, IL-18, GM-CSF, G-CSF, and any combination thereof.

The invention will be more fully understood by reference to thefollowing Examples. They should not, however, be construed as limitingthe scope of the invention. All citations throughout the disclosure arehereby expressly incorporated by reference.

EXAMPLES Example 1: Production, Identification, and Characterization ofAgonist Anti-TREM2 and Anti-DAP12 Antibodies Introduction

The amino acid sequence of the human TREM2 preprotein is set forth belowin SEQ ID NO: 1. Human TREM2 contains a signal peptide located at aminoresidues 1-18 of SEQ ID NO: 1. Human TREM2 contains an extracellularimmunoglobulin-like variable-type (IgV) domain located at amino residues29-112 of SEQ ID NO: 1; additional extracellular sequences located atamino residues 113-174 of SEQ ID NO: 1; a transmembrane domain locatedat amino residues 175-195 of SEQ ID NO: 1; and an intracellular domainlocated at amino residues 196-230 of SEQ ID NO: 1.

TREM2 amino acid sequence (SEQ ID NO: 1):

        10         20         30         40 MEPLRLLILL FVTELSGAHNTTVFQGVAGQ SLQVSCPYDS         50         60         70         80MKHWGRRKAW CRQLGEKGPC QRVVSTHNLW LLSFLRRWNG        90        100        110        120 STAITDDTLG GTLTITLRNLQPHDAGLYQC QSLHGSEADT        130        140        150        160LRKVLVEVLA DPLDHRDAGD LWFPGESESF EDAHVEHSIS       170        180        190        200 RSLLEGEIPF PPTSILLLLACIFLIKILAA SALWAAAWHG        210        220        230 QKPGTHPPSELDCGHDPGYQ LQTLPGLRDT

A known feature of human TREM2 is that the transmembrane domain containsa lysine (aa186) that can interact with an aspartic acid in DAP12, a keyadaptor protein that transduces signaling from TREM2, TREM1, and otherrelated IgV family members.

A BLAST analysis of human TREM2 identified 18 related homologues. Thesehomologues included the Natural Killer (NK) cell receptor NK-p44(NCTR2), the polymeric immunoglobulin receptor (pIgR), CD300E, CD300A,CD300C, and TREML1/TLT1. The closest homologue was identified as NCTR2,having similarity with TREM2 within the IgV domain (FIG. 1A). A BLASTanalysis also compared TREM proteins with other IgV family proteins(FIG. 1B).

TREM2 is also closely related to TREM1. An alignment of the amino acidsequences of TREM1 and TREM2 was generated by 2-way blast (FIG. 2A).This is limited to the IgV domain as well.

Agonistic antibodies for TREM1, NK-p44, and other members of this familyhave been previously described. Antibodies that bind the extracellulardomain of TREM2, particularly the IgV domain (amino acid residues 29-112of SEQ ID NO: 1) are generated using mouse hybridoma technology, phagedisplay technology, and yeast display technology. Antibodies are thenscreened for their ability to activate TREM2 signaling and functions incells and in a whole animal in vivo as described in Examples 2-39 below.

For example, agonist anti-TREM2 antibodies can be produced that targetthe IgV domain (amino acid residues 29-112). IgV domains bind totargets, and through multimerization of receptors, such as IgG itself orNKp44, lead to activation. Thus these domains are rational targets foragonistic antibodies. They are also highly divergent.

Agonist anti-TREM2 antibodies can also be produced that target aminoacid residues 99-115 of human TREM2. It is believed that amino acidresidues 99-115 correspond to a peptide that block binding of TREM2 toits endogenous target, as the corresponding peptide in mouse TREM1(amino acid residues 83-99) block binding of TREM1 to its endogenoustarget (Gibot et al., Infect. Immunity 2004). The mouse TREM1 peptide iscalled LP17 (LQVTDSGLYRCVIYHPP (SEQ ID NO: 414)). The equivalent regionin human TREM2 is located within the CD3 domain and is located at aminoacid residues 99-115 of SEQ ID NO: 1 (LQPHDAGLYQCQSLHG). Antibodies thatblock ligand binding could activate the receptors similar to the liganditself.

Another approach for predicting a relevant (e.g., agonistic) site withinthe human TREM2 protein is by targeting the sites where the mutationsare found in Alzheimer's disease (e.g., R47H), Polycystic lipomembranousosteodysplasia with sclerosing leukoencephalopathy (PLOSL), orNasu-Hakola disease. Also relevant is the site of the major mutationsassociated with human disease, which are generally found within the IgVdomain.

The crystal structures of the TREM2-related structures of TREM1 (Kelker,M S et al., J Mol Biol, 2004. 344(5): p. 1175-81; Kelker, M S et al., JMol Biol, 2004. 342(4): p. 1237-48; and Radaev, S et al., Structure,2003. 11(12): p. 1527-35), TLT1 (Gattis, J L et al., J Biol Chem, 2006.281(19): p. 13396-403), and NKp44 have been described, and thusstructural regions/features are identified within the IgV domain thatare particularly likely to play a central role in interacting withnatural agonists. These studies support the belief that thecomplementary determining regions (CDR1, CDR2, CDR3) play a major rolein ligand binding. TREM1 has been reported to be either monomeric(Gattis, J L et al., J Biol Chem, 2006. 281(19): p. 13396-403) ordimeric (Radaev, S et al., Structure, 2003. 11(12): p. 1527-35) in vitrounder cell-free conditions, but its oligomeric state in vivo remainsunclear, as well as that of TREM2.

The amino acid sequence of human DAP12 is set forth below as SEQ ID NO:2:

        10         20         30         40 MGGLEPCSRL LLLPLLLAVSGLRPVQAQAQ SDCSCSTVSP         50         60         70         80GVLAGIVMGD LVLTVLIALA VYFLGRLVPR GRGAAEAATR        90        100        110 KQRITETESP YQELQGQRSD VYSDLNTQRP YYK

DAP12 is a single-pass type I membrane protein. It contains anextracellular domain located at amino acid residues 22-40 of human DAP12(SEQ ID NO: 2); a transmembrane domain located at amino acid residues41-61 of human DAP12 (SEQ ID NO: 2); and an intracellular domain locatedat amino acid residues 62-113 of human DAP12 (SEQ ID NO: 2). Theimmunoreceptor tyrosine-based activation motif (ITAM) domain of DAP12 islocated at amino acid residues 80-118 of human DAP12 (SEQ ID NO: 2). Anaspartic acid in DAP12 interacts with the transmembrane domain of humanTREM2 containing a lysine at amino acid residue 186, and transducessignaling from TREM2, TREM1, and other related IgV family memberproteins.

Agonist anti-DAP12 antibodies can be produced that target amino acidresidues 22-40 of the human DAP12. It is believed that DAP12 is adisulfide-bonded dimer, associating with TREM2, and that dimerizingDAP12 with an antibody against the extra cellular domain encompassingamino acid residues 22-40 will activate one or more TREM2 and/or DAP12activities.

The studies discussed herein describe the generation of agonistantibodies that bind TREM2. Antibodies were screened for binding toTREM2 expressing cells and for their ability to activate TREM2 signalingand functionality.

Results

Anti-TREM2 Antibody Production

Antibodies that bind the extracellular domain of TREM2, particularly theIgV domain (amino acid residues 29-112 of SEQ ID NO: 1) were generatedusing the following procedure. Eight naïve human synthetic yeastlibraries each of ˜10⁹ diversity were designed, generated, andpropagated as described previously (see, e.g., WO2009036379;WO2010105256; WO2012009568; Xu et al., (2013) Protein. Eng. Des. Sel.26(10):663-670). The ADIMAB yeast-based antibody discovery platform usedherein allowed for the identification of fully human, full-length,monoclonal IgG1 antibodies with broad epitopic coverage. The ADIMAByeast is engineered to transport high quality, whole IgGs through thesecretory pathway, and then present them on the surface or secrete themdirectly into the medium.

For the first rounds of selection, a magnetic bead sorting techniqueutilizing the Miltenyi MACs system was performed, as previouslydescribed (Siegel et al., (2004) J. Immunol. Methods 286(1-2):141-53).Briefly, yeast cells (˜10¹⁰ cells/library) were incubated with 3 ml of200 nM biotinylated TREM2 antigen or 10 nM biotinylated TREM2-Fc fusionantigen for 15 min at room temperature in FACS wash buffer PBS with 0.1%BSA. Biotinylations were performed using the EZ-LinkSulfo-NHS-Biotinylation Kit (Thermo Scientific, Cat #21425). Afterwashing once with 50 ml ice-cold wash buffer, the cell pellet wasresuspended in 40 mL wash buffer, and 500 μl Streptavidin MicroBeads(Miltenyi Biotec, Bergisch Gladbach, Germany. Cat #130-048-101) wereadded to the yeast and incubated for 15 min at 4° C. Next, the yeastwere pelleted, resuspended in 5 mL wash buffer, and loaded onto a MACSLS column (Miltenyi Biotec, Bergisch Gladbach, Germany.Cat.#130-042-401). After the 5 mL was loaded, the column was washed 3times with 3 ml FACS wash buffer. The column was then removed from themagnetic field, and the yeast were eluted with 5 mL of growth media andthen grown overnight. The following three rounds of sorting wereperformed using flow cytometry. Approximately 1×10⁸ yeast were pelleted,washed three times with wash buffer, and incubated with 200 nM, 100, or10 biotinylated TREM2 for 10 min at room temperature respectively. Yeastwere then washed twice and stained with goat anti-human F(ab′)₂kappa-FITC diluted 1:100 (Southern Biotech, Birmingham, Ala.,Cat#2062-02) and either streptavidin-Alexa Fluor 633 (Life Technologies,Grand Island, N.Y., Cat # S21375) diluted 1:500 orExtravidin-phycoerthyrin (Sigma-Aldrich, St Louis, Cat # E4011) diluted1:50 secondary reagents for 15 min at 4° C. After washing twice withice-cold wash buffer, the cell pellets were resuspended in 0.4 mL washbuffer and transferred to strainer-capped sort tubes. Sorting wasperformed using a FACS ARIA sorter (BD Biosciences) and sort gates weredetermined to select only TREM2 binding clones for two rounds and thethird round was a negative sort to decrease reagent binders. After thefinal round of sorting, yeast were plated and individual colonies werepicked for characterization.

Yeast clones were grown to saturation and then induced for 48 h at 30°C. with shaking. After induction, yeast cells were pelleted and thesupernatants were harvested for purification. IgGs were purified using aProtein A column and eluted with acetic acid, pH 2.0. Fab fragments weregenerated by papain digestion and purified over KappaSelect (GEHealthcare LifeSciences, Cat #17-5458-01). Two antibodies (Ab21 andAb52) were selected for further analysis.

Heavy Chain and Light Chain Variable Domain Sequences of Antibodies Ab21and Ab52

Using standard techniques, the amino acid sequences encoding the heavychain variable (FIG. 2B) and the light chain variable (FIG. 2C) domainof antibody Ab21 and antibody Ab52 were determined.

The Kabat CDR sequences of antibody Ab21 and antibody Ab52 are set forthin Table 1.

The amino acid sequence of the heavy chain variable domain of antibodyAb21 is: EVQLVQSCAEVKKPGESLKISCKGSGYSFTTYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARAGHYDGGHLGMDVWGQGT TVTVSS (SEQ IDNO:410), and the amino acid sequence of the light chain variable domainof antibody 21 is:

(SEQ ID NO: 411) EIVMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASNRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQDDSAPYTFG GGTKVEIK.

The amino acid sequence of the heavy chain variable domain of antibodyAb52 is: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREADDSSGYPLGLDVWGQG TMVTVSS (SEQID NO:412), and the amino acid sequence of the light chain variabledomain of antibody 52 is:

(SEQ ID NO: 413) EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQVNSLPPTFGG GTKVEIK.

TABLE 1A Kabat heavy chain CDR sequences Antibody Name CDR H1 CDR H2 CDRH3 Ab21 YSFTTYWIG IIYPGDSDTRYSPSFQG ARAGHYDGGHLGMDV (SEQ ID NO: 404)(SEQ ID NO: 405) (SEQ ID NO: 406) Ab52 YTFTSYYIH IINPSGGSTSYAQKFQGAREADDSSGYPLGLDV (SEQ ID NO: 398) (SEQ ID NO: 399) (SEQ ID NO: 400)

TABLE 1B Kabat lightchain CDR sequences Antibody Name CDR L1 CDR L2 CDRL3 Ab21 RASQSVSSSYLA GASNRAT QQDDSAPYT (SEQ ID NO: 407) (SEQ ID NO: 408)(SEQ ID NO: 409) Ab52 RASQSVSSNLA GASTRAT QQVNSLPPT (SEQ ID NO: 401)(SEQ ID NO: 402) (SEQ ID NO: 403)

Characterization of Ab21 and Ab52 Binding

Initial characterization of TREM2 antibodies involved determining theirability to bind TREM2 expressed on dendritic and other primary human ormouse immune cells. Cells were harvested, plated at 10⁵/ml in a 96 wellplate, washed, and incubated in 100 ul PBS containing 10-50 ug/ml Maband Fc blocking reagent for 1 hour in ice. Cells were then washed twiceand incubated in 100 ul PBS containing 5 ug/ml PE-conjugated secondaryantibody for 30 minutes in ice. Cells were washed twice in cold PBS andacquired on a BD FACS Canto. Data analysis and calculation of MFI valueswas performed with FlowJo (TreeStar) software version 10.0.7.

Antibodies Ab21, Ab52, Ab16, Ab20, Ab66, and Ab68 demonstrated bindingto a mouse cell line (BWZ T2) expressing recombinant mouse TREM2, asindicated by positive TREM2 antibody staining detected via FACS analysis(black outlined histograms) (FIG. 3A). Antibodies Ab21 and Ab52demonstrated antibody binding to WT (Trem+/+) bone marrow derived mousemacrophages (BMMac, mMac), but not to TREM2 deficient (TREM2−/−) mousemacrophages (BMMac, mMacs) (FIG. 3B). Antibodies Ab21 and Ab52demonstrated binding to both a human cell line (293) expressingrecombinant Human TREM2 (FIG. 4A) and to primary human dendritic cells(hDC) (FIG. 4B). Conversely, antibodies Ab43 and ab60 bound to a humancell line expressing recombinant human TREM2 (FIG. 4A), but did not bindto primary human dendritic cells (FIG. 4B).

Mean fluorescent intensities (MFI) values for cell types bound by TREM2antibodies Ab21 and Ab52 are listed in Table 4. Binding is compared tothe parental mouse cell line (mTREM2 cell line BWZ parental), primaryhuman cell line (hTREM2 Parental Cell line (293)), primary mousemacrophages deficient in TREM2 (mMacs KO MFI), and primary mousedendritic cells deficient in TREM2 (mDC KO MFI).

TABLE 4 TREM2 Antibody Binding to Human and Mouse Cells mTREM2 hTREM2Cell line mTREM2 Parental hTREM2 (BWZ- Cell line Cell line Cell linemMacs mMacs mDC mDC parental) (BWZ T2) (293) (293) KO WT KO WT hDC %Antibody MFI MFI MFI MFT MFI MFI MFI MFI positive Ab52 1021 15613 891411 73.3 174.0 203 663 69.1 Ab21 1036 13840 81 1884 64.7 111.0 187 46477.1

The binding affinity of each anti-TREM2 antibody was determined bymeasuring their K_(D) by ForteBio or MSD-SET. ForteBio affinitymeasurements were performed as previously described (Estep et al, (2013)MAbs 5(2):270-8). Briefly, ForteBio affinity measurements were performedby loading IgGs on-line onto AHQ sensors. Sensors were equilibratedoff-line in assay buffer for 30 min and then monitored on-line for 60seconds for baseline establishment. Sensors with loaded IgGs wereexposed to 100 nM antigen for 5 minutes, then transferred to assaybuffer for 5 min for off-rate measurement. Kinetics were analyzed usingthe 1:1 binding model.

Equilibrium affinity measurements were performed as previously described(Estep et al, (2013) MAbs 5(2):270-8). Solution equilibrium titrations(SET) were performed in PBS+0.1% IgG-Free BSA (PBSF) with antigen heldconstant at 50 pM and incubated with 3- to 5-fold serial dilutions ofantibody starting at 10 nM. Antibodies (20 nM in PBS) were coated ontostandard bind MSD-ECL plates overnight at 4° C. or at room temperaturefor 30 min. Plates were then blocked for 30 min with shaking at 700 rpm,followed by three washes with wash buffer (PBSF+0.05% Tween 20). SETsamples were applied and incubated on the plates for 150s with shakingat 700 rpm followed by one wash. Antigen captured on a plate wasdetected with 250 ng/mL sulfotag-labeled streptavidin in PBSF byincubation on the plate for 3 min. The plates were washed three timeswith wash buffer and then read on the MSD Sector Imager 2400 instrumentusing 1× Read Buffer T with surfactant. The percent free antigen wasplotted as a function of titrated antibody in Prism and fit to aquadratic equation to extract the K_(D). To improve throughput, liquidhandling robots were used throughout MSD-SET experiments, including SETsample preparation.

Table 5 lists values representing the binding affinity (K_(D)) ofantibodies Ab21 and Ab52 to a human TREM2 Fc fusion protein (hTREM2-Fc),a human monomeric His tagged TREM2 protein (hTREM2-HIS), and a mouseTREM2 Fc fusion protein (mTREM2-Fc).

TABLE 5 Binding affinity of TREM2 antibodies IgG K_(D) IgG K_(D) IgGK_(D) hTREM2-Fc hTREM2-HIS (M) mTREM2-Fc Antibody (M) Avid Monovalent(M) Avid Ab52 1.51E−09 5.75E−09 8.96E−11 Ab21 3.44E−10 1.14E−09 2.27E−10

Example 2: Normalization and Reduction of Toll-Like Receptor (TLR)Responses in Dendritic Cells by Agonistic TREM2, DAP12, and/orTREM2/DAP12 Bispecific Antibodies

Bone marrow-derived dendritic cells (BMDC) are stimulated by culturingwith TLR ligands, such as LPS, CpG DNA, and zymosan, for 16 h.Conditioned media is collected and ELISA assays are performed in orderto evaluate secretion of the cytokines IFN-a4, IFN-b, IL-6, IL-12 p70,and TNF. It is believed that BMDC cells that do not have active TREM2may secrete significantly more IL-12, p70, and TNF than BMDC cells thathave activated TREM2 after stimulation. It is further believed thatanti-TREM2 agonistic antibodies will reduce the expression levels ofIL-12, p70, and TNF. Bone marrow-derived dendritic cells from wild-typeand from TREM2-hetrozyous mice, which would have partially inactiveTREM2, will serve as positive controls for determining expression levelsof the cytokines IL-12, p70, and TNF, as well as their modulation byagonistic anti-TREM2, anti-DAP12, and/or TREM2/DAP12 bispecificantibodies.

Cytokine concentrations in the culture supernatants are determined usingmouse IFN-a4, IFN-b, IL-6, IL-12 p70, TNF, and IL-10 ELISA kits(eBioscience) and VeriKine Mouse IFN-b ELISA kit (PBL interferon source)according to manufacturer's protocol. Levels of mRNA for these cytokinesare also measured by Quantitative RT-PCR (qRT-PCR). Total RNA isprepared by using RNeasy plus mini kit (QIAGEN) is reverse-transcribedwith Superscript III Reverse Transcriptase (Invitrogen) using oligo dTprimer according to manufacturer's protocol. Quantitative PCR isperformed using the Power SYBR Green PCR Master Mix (Applied Biosystems)and 7900HT (Applied Biosystems) according to manufacturer's protocol.The sequences of IFN-a4, IFN-b, IL-6, IL-12 p70, and TNF primers are asdescribed. (e.g., Hamerman, J A, Eur. J. Immunol. 2012. 42: 176-185).

Example 3: Normalization and Reduction of the Ability of BMDCs to InduceAntigen-Specific T-Cell Proliferation by Agonistic TREM2, DAP12, and/orTEM2/DAP12 Bispecific Antibodies

It is believed that agonistic anti-TREM2, anti-DAP12, and/or TREM2/DAP12bispecific antibodies may reduce and normalize the ability of bonemarrow-derived dendritic cells (BMDC) to induce antigen-specific T-cellproliferation.

Ovalbumin (OVA)-specific T-cell response induced by BMDCs can bedetermined by CFSE dilution. BMDCs are isolated by MACS after 6 days ofculture and plated at 1×10⁴ cells per well of a round bottom 96 wellplate with OVA (2 or 0.5 mg/mL) and CpG DNA (100 or 25 nM) in thepresence of GM-CSF (10 ng/mL) for 4 h. CD4 T-cells from the spleen andlymph nodes of OT-II transgenic mice are isolated by using Dynal MouseCD4 Negative Isolation Kit (Invitrogen) and stained with CFSE (final 0.8mM). After 4 h of DC culture, 1×10⁵ CFSE-labeled CD4 OT-II T-cells areadded into each well and incubated for 72 h. After culturing, cells arestained with an anti-CD4 monoclonal antibody and flow cytometry isperformed to detect CFSE dilution of gated CD4 OT-II T-cells. Dataanalysis to calculate the percentage of divided and division index isperformed by Flowjo software (Treestar) (Eur. J. Immunol. 2012. 42:176-185).

Example 4: Normalization and Reduction of Toll-Like Receptor (TLR)Responses in Macrophages by Agonistic TREM2, DAP12, and/or TEM2/DAP12Bispecific Antibodies

Bone marrow-derived macrophages (BMDM) or primary peritoneal macrophageresponses are altered to TLR signaling by deficiency of TREM2 (Turnbull,I R et al., J Immunol 2006; 177:3520-3524). It is believed thatagonistic anti-TREM2, anti-DAP12, and/or TREM2/DAP12 bispecificantibodies may reduce and normalize TLR responses in macrophages.

To elicit primary macrophages, mice are treated with 1.5 ml of 2%thioglycollate medium by intraperitoneal injection, and cells are thenisolated by peritoneal lavage. To generate BMDM, total bone marrow iscultured in DMEM supplemented with 10% bovine calf serum, 5% horseserum, and 6 ng/ml recombinant human CSF-1 (R&D Systems). Cells arecultured for 5-6 days, and adherent cells are detached with 1 m MEDTA inPBS. Cells are stained with commercially available antibodies:anti-CD11b, anti-CD40, anti-GR1 (BD Pharmingen), and F4/80 (CaltagLaboratories).

BMDM are re-plated and allowed to adhere for 4 h at 37° C., and then TLRagonists, such as LPS (Salmonella abortus equi), zymosan (Saccharomycescerevisiae), and CpG 1826 DNA (purchased from e.g., Sigma-Aldrich) areadded. Cell culture supernatant is collected 24 h after stimulation andthe levels of IFN-a4, IFN-b, IL-6, IL-12 p70, and TNF cytokines aremeasured by ELISA or by cytometric bead array (BD Biosciences mouseinflammation kit).

Example 5: Induction of the Anti-Inflammatory Cytokine IL-10 in BoneMarrow-Derived Myeloid Precursor Cells by Agonistic TREM2, DAP12, and/orTEM2/DAP12 Bispecific Antibodies

It is believed that bone marrow-derived myeloid precursor cells may showan increase in the anti-inflammatory cytokine IL-10 following treatmentwith agonistic anti-TREM2, anti-DAP12, and/or TREM2/DAP12 bispecificantibodies and stimulation with 100 ng/ml LPS (Sigma), by co-culturingwith apoptotic cells, or by a similar stimulus.

Isolation of bone marrow-derived myeloid precursor cells is performed asfollows. Bone marrow cells are isolated from adult 6-8 week-old femaleC57BL/6 mice (Charles River, Sulzfeld, Germany) from the medullarycavities of the tibia and femur of the hind limbs. Removal oferythrocytes is performed by lysis with hypotonic solution. Cells arecultured in DMEM medium (Invitrogen) containing 10% fetal calf serum(Pan Biotech) and 10 ng/ml of GM-CSF (R&D Systems) in 75 cm² cultureflasks (Greiner Bio-One). After 24 h, non-adherent cells are collectedand re-seeded in fresh 75 cm² culture flasks. Medium is changed after 5d and cells are cultured for an additional 10-11 d. The remaining cellsare bone marrow-derived myeloid precursor cells, and are transduced withTREM2 virus. The transduced cells are then examined for the level ofIL-10 in conditioned media in both the presence and absence ofanti-TREM2 agonistic antibodies and LPS. Supernatant is collected after24 h, and the level of IL-10 released from the cells is determined byIL-10 ELISA according to manufacturer's instructions (QuantikineM mouseIL-10, R&D Systems) (JEM (2005), 201; 647-657; and PLoS Medicine (2004),4 | Issue 4 | e124).

Example 6: Induction of Phagocytosis of Apoptotic Neurons, Nerve TissueDebris, Non-Nerve Tissue Debris, Bacteria, Other Foreign Bodies, andDisease-Causing Proteins in Cells from the Myeloid Lineage by AgonisticTREM2, DAP12, and/or TEM2/DAP12 Bispecific Antibodies

It is believed that agonistic anti-TREM2, anti-DAP12, and/or TREM2/DAP12bispecific antibodies may induce phagocytosis of apoptotic neurons,nerve tissue debris, non-nerve tissue debris, bacteria, other foreignbodies, and disease-causing proteins, such as A beta peptide, alphasynuclain protein, Tau protein, TDP-43 protein, prion protein, andhuntingtin protein, in cells from the myeloid lineage, such as monocytesand microglia.

Monocytes are isolated from peripheral blood that is collected fromadult C57BL/6 mice. Hypotonic lysis buffer depletes crythrocytes. Cellsare plated on culture dishes in RPMI medium (Invitrogen) containing 10%fetal calf serum (Pan Biotech). Cells are cultured for several hours at37° C. in 10% CO₂. After trypsinization, adherent cells are collectedand used for phagocytosis experiments.

Microglial cells are prepared from the brains of post-natal day 3 to 5(P3 to P5) C57BL/6 mice. In brief, meninges are removed mechanically,and the cells are dissociated by trituration and cultured in basalmedium (BME; GIBCO BRL) supplemented with 10% FCS (PAN Biotech GmbH), 1%glucose (Sigma-Aldrich), 1% L-glutamine (GIBCO BRL), and 1%penicillin/streptomycin (GIBCO BRL), for 14 d to form a confluent glialmonolayer. To collect microglial cells, the cultures are shaken on arotary shaker (200 rpm) for 2 h. The attached astrocytes are used forimmunohistochemistry. The detached microglial cells are seeded in normalculture dishes for 1 h, and then all non-adherent cells are removed anddiscarded. Purity of the isolated microglial cells is about 95% asdetermined by flow cytometry analysis with antibody directed againstCD11b (BD Biosciences). Microglial cells are cultured in basal medium.

Oligodendrocytes (i. e., neurons) and neuron-enriched cells are preparedfrom the brain of C57BL/6 mouse embryos (E15-16). In brief, brain tissueis isolated and mechanically dispersed and seeded in culture dishespre-coated with 0.01 mg/ml poly-L-ornithin (Sigma-Aldrich) and 10 μg/mllaminin (Sigma-Aldrich). Cells are cultured in neuronal condition medium(BME; GIBCO BRL) supplemented with 2% B-27 supplement (GIBCO BRL), 1%glucose (Sigma-Aldrich), and 1% FCS (PAN Biotech GmbH). Cells arecultured for 5-10 d to obtain morphologically mature oligodendrocytes.

To conduct phagocytosis assays of apoptotic neurons, nerve tissuedebris, non-nerve tissue debris, bacteria, other foreign bodies, anddisease-causing proteins, microglia are transduced with sh-TREM2 RNA,sh-control RNA, wTREM2, GFP1 control, mtDAP12-GFP, and GFP2 controlvector. After transduction, microglia are cultured for 72 h to achieveeffective knockdown of TREM2 by RNA interference. Neurons are culturedfor 5-10 d, and okadaic acid is then added at the final concentration of30 nM for 3 h to induce apoptosis. Neuronal cell membranes are labeledwith CellTracker CM-DiI membrane dye (Molecular Probes). Afterincubation, apoptotic neurons or other targets of phagocytosis arewashed two times and added to the transduced microglial culture at aneffector/target ratio of 1:20. At 1 and 24 h after addition of apoptoticneurons, the number of microglia having phagocytosed neuronal cellmembranes is counted under a confocal fluorescence microscope (Leica).Apoptotic cells are counted in three different areas at a magnificationof 60. The amount of phagocytosis is confirmed by flow cytometry.Moreover, 24, 48, or 72 h after the addition of apoptotic neurons, cellsare collected and used for RT-PCR of cytokines.

To conduct microsphere bead or bacterial phagocytosis assay, microgliaare transduced with a TREM2 expression vector or a GFP control vector.Cells are then treated with anti-TREM2 agonistic antibodies. After 24 h,1.00 μm of red fluorescent microsphere beads (Fluoresbrite PolychromaticRed Mi-crospheres; Polysciences Inc.) or, fluorescent labeled bacteriaare added for 1 h. Phagocytosis of microsphere beads or, fluorescentlabeled bacteria, by microglia is analyzed by fluorescence microscopy.Furthermore, microglia are collected from the culture plates andanalyzed by flow cytometry. The percentage of microglia havingphagocytosed beads is determined. Because phagocytosis varies from oneexperiment to the other, the relative change in phagocytosis is alsodetermined. Data are shown as the relative change in phagocytosisbetween microglia cultured with agonistic antibodies and controlantibody.

To conduct RT-PCR for analysis of inflammatory gene transcripts,microglia are transduced with a TREM2 vector or a GFP1 control vector.Cells are then cultured on dishes and treated with anti-TREM2 agonisticantibodies. After 24, 48, and 72 h, RNA is isolated from microglia usingan RNeasy Mini Kit (QIAGEN). RNA is also collected from microglia thathave been transduced with sh-TREM2 RNA, sh-control RNA, wTREM2, GFP2,mtDAP12-GFP, and GFP1 vector and co-cultured with apoptotic neurons for48 h.

Reverse transcription of RNA is then performed. Quantitative RT-PCR bySYBR Green is performed on an ABI Prism 5700 Sequence Detection System(PerkinElmer). Amplification of GAPDH is used for sample normalization.The amplification protocol followed the GeneAmp 5700 Sequence DetectionSystem Software (version 1.3). For detection of GAPDH, TNF-alpha, IL-1,NOS2, and TGF-beta transcripts, the following forward and reverseprimers were used at final concentrations of 200 nM:

GAPDH forward primer: (SEQ ID NO: 416) 5′-CTCCACTCACGGCAAATTCAA-3′, andGAPDH reverse primer: (SEQ ID NO: 417) 5′-GATGACAAGCTTCCCATTCTCG-3′;TNF-α forward primer: (SEQ ID NO: 418) 5′-CCGTCAGCCGATTTGCTATCT-3′, andTNF-α reverse primer: (SEQ ID NO: 419) 5′-ACGGCAGAGAGGAGGTTGACTT-3′;IL-1α forward primer: (SEQ ID NO: 420) 5′-ACAA-CAAAAAAGCCTCGTGCTG-3′,and IL-1α reverse primer: (SEQ ID NO: 421) 5′-CCATTGAGGTGGAGAGCTTTCA-3′;NOS2 forward primer: (SEQ ID NO: 422) 5′-GGCAAACCCAAGGTCTACGTTC-3′, NOS2reverse primer: (SEQ ID NO: 423) 5′-TACCTCATTGGCCAGCTGCTT-3′; and TGF-β1forward primer: (SEQ ID NO: 424) 5′-AGGACCTGGGTTGGAAGTGG-3′, andTGF-β1reverse primer: (SEQ ID NO: 425) 5′-AGTTGGCATGGTAGCCCTTG-3′.

To conduct amyloid phagocytosis assay, HiLyteFluor™ 647(Anaspec)-Abeta-(1-40) was resuspended in Tris/EDTA (pH 8.2) at 20 mMand then incubated in the dark for 3 d at 37° C. to promote aggregation.Microglial cells are pretreated in low serum (0.5% FBS supplemented withinsulin), LPS (50 ng/ml), IFNc (100 units/ml), and anti-TREM2 agonisticantibodies for 24 h prior to the addition of aggregated fluorescentlylabeled a beta peptide. Amyloid phagocytosis and surface expression ofTREM2 are determined by flow cytometric analysis 5 h post-addition of100 nM aggregated HiLyteFluor™ 647-Ab-(1-40) (ASN NEURO (2010) 2(3):157-170). Phagocytosis of other disease-causing proteins is conducted ina similar manner.

Example 7: Induction of ERK Activation by Agonistic TREM2, DAP12, and/orTEM2/DAP12 Bispecific Antibodies

It is believed that agonistic anti-TREM2, anti-DAP12, and/or TREM2/DAP12bispecific antibodies may induce ERK activation.

Microglia are transduced with a TREM2 vector, and 2×10⁵ cells areexposed to agonistic anti-TREM2, anti-DAP12, and/or TREM2/DAP12bispecific antibodies for 1 h. After stimulation, cells are lysed inreducing sample buffer for Western blot analysis. Phosphorylation of ERKand total amount of ERK are determined by immuno-detection withanti-phospho-ERK and anti-ERK antibodies, respectively (both from CellSignaling Technology) by Western blot analysis (JEM (2005), 201,647-657).

Example 8: Induction of CCR7 and Migration Toward CCL19 and CCL21 inMicroglia, Macrophages, and Dendritic Cells by Agonistic TREM2, DAP12,and/or TEM2/DAP12 Bispecific Antibodies

It is believed that agonistic anti-TREM2, anti-DAP12, and/or TREM2/DAP12bispecific antibodies may induce CCR7 and migration toward CCL19 andCCL21 in microglial cells, macrophages, and dendritic cells.

Microglial cells are transduced with a TREM2 vector or a GFP1 controlvector. The transduced microglial cells are then either cultured withagonistic anti-TREM2, anti-DAP12, and/or TREM2/DAP12 bispecificantibodies, or with a control antibody. Cells are collected after 72 h,immuno-labeled with CCR7 specific anti-bodies, and analyzed by flowcytometry.

To determine any functional consequences of increased CCR7 expression, achemotaxis assay is performed. Microglial cells are stimulated via TREM2with the agonistic anti-TREM2, anti-DAP12, and/or TREM2/DAP12 bispecificantibodies and placed in a two-chamber system. The number of microglialcells migrating toward the chemokine ligands CCL19 and CCL21 isquantified (JEM (2005), 201, 647-657).

For the chemotaxis assay, microglial cells are exposed to the agonisticanti-TREM2, anti-DAP12, and/or TREM2/DAP12 bispecific antibodies andtreated with 1 μg/ml LPS. Microglia are transferred into the upperchamber of a transwell system (3 μm pore filter; Millipore) containing450 μl medium with 100 ng/ml CCL19 or CCL21 (both from PeproTech) in thelower chamber. After a 1 h incubation period, the number of microglialcells that have migrated to the lower chamber is counted in threeindependent areas by microscopy (JEM (2005), 201, 647-657).

Example 9: Induction of F-Actin in Microglia, Macrophages, and DendriticCells by Agonistic TREM2, DAP12, and/or TEM2/DAP12 Bispecific Antibodies

It is believed that agonistic anti-TREM2, anti-DAP12, and/or TREM2/DAP12bispecific antibodies may induce F-actin in microglial cells,macrophages, and dendritic cells.

Microglia and other cells of interest that are transduced with TREM2 orthat express TREM2 are added to culture plates and then exposed toagonistic anti-TREM2, anti-DAP12, and/or TREM2/DAP12 bispecificantibodies, or a control antibody. Cells are fixed, blocked, and thenstained with Alexa Fluor 546-conjugated phalloidin (Molecular Probes)after 1 h and F-actin is labeled with a fluorescence dye. Images arecollected by confocal laser scanning microscopy with a 40× objectivelens (Leica). (JEM (2005), 201, 647-657).

Example 10: Induction of Ostcoclast Production and Increased Rate ofOstcoclastoecnesis by Agonistic TREM2, DAP12, and/or TEM2/DAP12Bispecific Antibodies

It is believed that agonistic anti-TREM2, anti-DAP12, and/or TREM2/DAP12bispecific antibodies may induce osteoclast production and increase therate of osteoclastogenesis.

RAW264.7 cells that make osteoclasts or bone marrow-derivedmonocyte/macrophage (BMM) precursor cells are maintained in RPMI-1640medium (Mediatech), or another appropriate medium, supplemented with 10%FBS (Atlantic Biologics, Atlanta, Ga., USA) andpenicillin-streptomycin-glutamine (Mediatech). TREM2B cDNA with a FLAGepitope added to the N terminus is inserted into the retroviral vectorpMXpie upstream of an IRES, followed by an eGFP cDNA sequence. Cells aretransfected with pMXpie-FLAG TREM2B, using Fugene 6 (Roche) according tomanufacturer's protocol. Cells are selected in puromycin (Sigma) at 2μg/ml. Stable puromycin-resistant clones are screened for anti-FLAG M2monoclonal antibody (Sigma) binding by using flow cytometry, and thensubcloned and maintained on puromycin selection media.

RAW264.7 cells expressing TREM2B are seeded in 96-well plates with 3000cells/well in alpha-MEM medium supplemented with 10% FBS,penicillin-streptomycin-glutamine, 50 ng/ml RANKL, and 20 ng/ml M-CSF.The medium is changed every 3 days, exposed to anti-TREM2 agonisticantibodies and the number of multinucleated (at least three nuclei)TRACP⁺ osteoclasts are counted and scored by light microscopy. Todetermine complexity and size, osteoclasts are counted by number ofnuclei (>10 or 3-10 nuclei). The surface area of osteoclasts is alsomeasured by using Image J software (NIH). In addition, expression levelsof osteoclasts genes are determined. Total RNA is extracted fromosteoclastogenic cultures at different time points using TRIzol reagent(Invitrogen). After first-strand cDNA synthesis using a SuperScript IIIkit (Invitrogen), real-time quantitative PCR reactions are performed forNfatc1, Acp5, Ctsk, Calcr, and Ccnd1. Relative quantification of targetmRNA expression is calculated and normalized to the expression ofcyclophilin and expressed as (mRNA of the target gene/mRNA ofcyclophilin) 3×10⁶. (J. OF BONE AND MINERAL RESEARCH (2006), 21,237-245; J Immunol 2012; 188:2612-2621).

Example 11: In Vivo Protection from EAE and Cuprizone in a Whole Animal

Adult 7-9 week-old female C57BL/6 mice (obtained from Charles RiverLaboratories) are injected in the tail base bilaterally with 200 μl ofan innoculum containing 100 μg of myelin oligodendrocyte glycoproteinpeptide 35-55 (amino acids MEVGWYRSPFSRVVHLYRNGK (SEQ ID NO: 415);Seqlab) and 1 mg of Mycobacterium tuberculosis H37 Ra (Difco) inincomplete Freund adjuvant (Difco). Pertussis toxin (200 ng; ListBio-logical Laboratories) is injected at day 0 and at day 2 afterimmunization. Clinical signs are scored as follows: 0, no clinicalsigns; 1, complete limp tail; 2, complete limp tail and abnormal gait;3, one hind-limb paraparesis; 4, complete hindlimb paraparesis; and 5,fore- and hind-limb paralysis or moribund.

Only mice having disease onset (clinical score of 1 or more) at day 14are used for experiments. Agonistic anti-TREM2, anti-DAP12, and/orTREM2/DAP12 bispecific antibodies are injected intraperitoneally orintravenously in EAE-diseased mice at the day of the first clinicalsymptoms or at any other desired time (PLoS Med (2007) 4(4): e124).

Young or aged wild-type (WT) mice are fed a standard diet (Harlan)containing 0.2% cuprizone (CPZ) powdered oxalicbis(cyclohexylidenehydrazide) (Sigma-Aldrich) for 4, 6 or 12 weeks. ForHistological and immunohistochemical analyses brains are removed aftermouse perfusion with 4% paraformaldehyde (PFA), fixed in 4% PFA for 24h, followed by immersion in 30% sucrose for 24-48 h. To evaluate myelinintegrity and damage, as well as cell proliferation and inflammationsections or mouse brain are stained with anti-MBP (1:100; Abcam,ab7349), -dMBP (1:2000; Millipore, ab5864), -β APP (1:100; Invitrogen,51-2700), -SMI-31 (1:1000; Covance, smi-31R), -Iba1 (1:600; Wako,019-19741), -BrdU (1:250; Abcam, ab1893), -GFAP (1:200; Invitrogen,13-0300), -iNOS (1:100; BD Pharmingen, 610329), -LPL (1:400, from Dr. G.Olivecrona) and -MHC II (1:100; BD Pharmingen, 553549). For behavioraleffects of the antibodies, mice are analyzed for locomotor activityusing transparent polystyrene enclosures and computerized photobeaminstrumentation. General activity variables (total ambulations, verticalrearings), along with indices of emotionality including time spent,distance traveled and entries, are analyzed. A battery of sensorimotortests is performed to assess balance (ledge and platform), strength(inverted screen), coordination (pole and inclined screens) andinitiation of movement (walking initiation). Motor coordination andbalance are studied using a rotarod protocol (Cantoni et al., ActaNeuropathol (2015)129(3):429-47).

Example 12: Characterization of the Therapeutic Use of Agonistic TREM2,DAP12, and/or TEM2/DAP12 Bispecific Antibodies in Established AnimalModels of Traumatic Brain Injury

The therapeutic utility of agonistic anti-TREM2, anti-DAP12, and/orTREM2/DAP12 bispecific antibodies is tested in established animal modelsof traumatic brain injury (Tanaka, Y et al. (2013) Neuroscience 23149-60).

For example, a model of traumatic brain injury that induces theactivation of microglia and astrocytes is used. Eight or nine week-oldmale C57BL/6J WT mice or progranulin heterozygous mice are used(purchased from Charles River Laboratories or Jackson Laboratories).Mice are anesthetized by intraperitoneal administration of xylazinehydrochloride (8 mg/kg) and chloral hydrate (300 mg/kg) dissolved insterile saline, and subsequently placed in a stereotaxic apparatus(Narishige, Tokyo, Japan). An incision is made in the scalp and thecranium is exposed. The periosteum is cleaned from the skull, a hole isdrilled over the right cerebral hemisphere with a dental drill, and theduramater is removed with a needle tip. A stainless steel cannula, witha 0.5 mm outer diameter, is used to make a longitudinal stab wound inthe right hemisphere. The cannula is positioned at 1.3 mm lateral to themidline, and 1 mm posterior to bregma, and introduced into the brainuntil the tip reaches a depth of 2 mm. The cannula is then shifted 2 mmcaudally (bregma 3 mm), and then shifts back 2 mm rostrally to itsinitial position. Finally, the cannula is removed from the brain, andthe scalp wound is sutured. Mice are then treated with agonisticanti-TREM2, anti-DAP12, and/or TREM2/DAP12 bispecific antibodiesaccording to standard procedures and then analyzed by histology andimmunofluorescence staining and behavioral tests.

Example 13: Characterization of Therapeutic Use of Agonistic TREM2,DAP12, and/or TEM2/DAP12 Bispecific Antibodies in a Model ofNeuro-Inflammation and Neuron Loss Following Toxin-Induced Injury

The therapeutic utility of agonistic anti-TREM2, anti-DAP12, and/orTREM2/DAP12 bispecific antibodies is tested in a model ofneuro-inflammation and neuron loss following toxin-induced injury(Martens, L H et al., (2012) The Journal of Clinical Investigation, 122,3955).

Three-month-old mice are treated with 4 intraperitoneal injections ofMPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) per day for 2 days(4 μg/g body weight) (Sigma-Aldrich) or PBS. Mice are treated withagonistic anti-TREM2, anti-DAP12, and/or TREM2/DAP12 bispecificantibodies according to standard protocols and then analyzed usingStereological counting to quantify dopamine neurons and microglia in thesubstantia nigra pars compacta (SNpc), as described.

Example 14: Characterization of the Therapeutic Use of Agonistic TREM2,DAP12, and/or TEM2/DAP12 Bispecific Antibodies in Animal Models ofAging, Seizures, Spinal Cord Injury, Retinal Dystrophy, FrontotemporalDementia, and Alzheimer's Disease

The therapeutic utility of agonistic anti-TREM2, anti-DAP12, and/orTREM2/DAP12 bispecific antibodies is tested in animal models for aging,seizures, spinal cord injury, retinal dystrophy, frontotemporaldementia, and Alzheimer's disease, as previously described (e.g.,Beattie, M S et al., (2002) Neuron 36, 375-386; Volosin, M et al.,(2006) J. Neurosci. 26, 7756-7766; Nykjaer, A et al., (2005) Curr. Opin.Neurobiol. 15, 49-57; Jansen, P et al., (2007) Nat. Neurosci. 10,1449-1457; Volosin, M et al., (2008) J. Neurosci. 28, 9870-9879;Fahnestock, M et al., (2001) Mol. Cell Neurosci. 18, 210-220; Nakamura,K et al., (2007) Cell Death. Differ. 14, 1552-1554; Yune, T et al.,(2007) Brain Res. 1183, 32-42; Wei, Y et al., (2007) Neurosci. Lett.429, 169-174; Provenzano, M J et al., (2008) Laryngoscope 118, 87-93;Nykjaer, A et al., (2004) Nature 427, 843-848; Harrington, A W et al.,(2004) Proc. Natl. Acad. Sci. U.S.A. 101, 6226-6230; Teng, H K et al.,(2005) J. Neurosci. 25, 5455-5463; Jansen, P et al., (2007) Nat.Neurosci. 10, 1449-1457; Volosin, M et al., (2008) J. Neurosci. 28,9870-9879; Fan, Y J et al., (2008) Eur. J. Neurosci. 27, 2380-2390;Al-Shawi, R et al., (2008) Eur. J. Neurosci. 27, 2103-2114; and Yano, Het al., (2009) J. Neurosci. 29, 14790-14802).

Example 15: Characterization of the Therapeutic Use of Agonistic TREM2,DAP12, and/or TEM2/DAP12 Bispecific Antibodies in Models ofAtherosclerosis

The therapeutic utility of agonistic anti-TREM2, anti-DAP12, and/orTREM2/DAP12 bispecific antibodies is tested in models ofatherosclerosis, as previously described (e.g., Lance, A et al., (2011)Diabetes, 60, 2285; and Kjolby, M et al., (2012) Cell Metabolism 12,213-223).

Example 16: Characterization of the Therapeutic Use of Agonistic TREM2,DAP12, and/or TEM2/DAP12 Bispecific Antibodies in a Model of Infection

The therapeutic utility of agonistic anti-TREM2, anti-DAP12, and/orTREM2/DAP12 bispecific antibodies is tested in a model of infection. Forexample, Listeria monocytogenes or other infection in normal mice orprogranulin heterozygous mice can be used, as previously described(e.g., Yin, F et al., (2009) J. Exp. Med, 207, 117-128).

Example 17: Characterization of the Therapeutic Use of Agonistic TREM2,DAP12, and/or TEM2/DAP12 Bispecific Antibodies in a Model ofInflammatory Diseases

The therapeutic utility of agonistic anti-TREM2, anti-DAP12, and/orTREM2/DAP12 bispecific antibodies is tested in a model of inflammatorydiseases. For example rheumatoid arthritis or in an established model ofanother inflammatory disease (Mizoguchi (2012) Prog Mol Biol TranslSci., 105:263-320; and Asquith et al., (2009) Eur J Immunol. 39:2040-4).

Example 18: Screening for Anti-TREM2, Anti-DAP12, and/or TEM2/DAP12Bispecific Antibodies that Induce Phosphorylation of DAP12, ERK, and AKTwhich Indicate Activation of the PI3K Pathway

Cells (J774, RAW 264.7, BMM cells, or osteoclasts) are removed fromtissue culture dishes with PBS-EDTA, washed with PBS, and counted. J774(40×10⁶) or RAW 264.7 cells (10×10⁶ BMM or osteoclasts) are incubatedwith an anti-TREM2, anti-DAP12, and/or TREM2/DAP12 bispecific antibodyor with an isotype-matched control antibody at 1 μg/10⁶ cells for 20 minon ice or under other conditions. Cells are lysed in ice-coldradioimmunoprecipitation assay (RIPA) buffer [50 mM tris-HCl (pH 7.4),150 mM NaCl, 1 mM EDTA, 1% Triton-100, 1 mM NaF, 1 mMphenylmethylsulfonyl fluoride, 1 mM Na VO, 0.25% sodium deoxycholate,aprotinin (1 μg/ml), leupeptin (1 μg/ml), pepstatin (1 μg/ml)] for 20min followed by centrifugation at 16,000 g for 10 min at 4° C. to removeinsoluble materials. The resulting supernatant is subjected toimmunoprecipitation reactions with the indicated antibodies (DAP12, ERK,or AKT) and protein A- or protein G-agarose (Sigma). The beads areextensively washed with RIPA buffer and the proteins are separated bySDS-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins are thentransferred to nitrocellulose membranes by Western blotting, incubatedwith the appropriate antibodies (antibodies that specifically recognizethe phosphorylated form of DAP12, ERK, or AKT) and visualized with theenhanced chemiluminescence (ECL) system (Pierce), as described (e.g.,Peng et al., (2010) Sci Signal., 3(122): ra38).

Example 19: Screening for Anti-TREM2, Anti-DAP12, and/or TEM2/DAP12Bispecific Antibodies that Induce Calcium Flux

BMM cells are washed twice with HEPES-containing buffer [20 mM HEPES (pH7.3), 120 mM NaCl, 1 mM CaCl, 1 mM MgCl, 5 mM KCl, glucose (1 mg/ml),bovine serum albumin (1 mg/ml)] followed by incubation in 0.05% PluronicF-127 (Invitrogen) and 1 μM Indo-1 AM (Invitrogen) for 20 min at 37° C.Cells are washed twice with HEPES buffer and are then stimulated with ananti-TREM2, anti-DAP12, and/or TREM2/DAP12 bispecific antibody (16μg/ml) or with a control antibody (16 μg/ml) and monitored byspectrophotometer (PTL Photon Technology International). The Indo-1fluorescence emission is converted to calcium (Ca²⁺) according tomanufacturer's instructions (e.g., Peng et al., (2010) Sci Signal.,3(122): ra38).

Example 20: Screening for Anti-TREM2, Anti-DAP12, and/or TEM2/DAP12Bispecific Antibodies that Prevent Apoptosis

Mature osteoclast cell cultures are differentiated in 24-well disheswith RANKL and M-CSF. After 4 days, complete medium is substituted withserum-free medium to induce apoptosis. Cells are treated with RANKL,PBS, and an anti-TREM2, anti-DAP12, and/or TREM2/DAP12 bispecificantibody, or an isotype-matched control antibody, during the overnightserum starvation. Cells are fixed in 1% paraformaldehyde and stainedwith a TUNEL-based kit (Millipore Corporation) according tomanufacturer's instructions. Apoptotic nuclei are counted with a NikonTE2000-E microscope with 20× magnification. Results are expressed as thepercentage of apoptotic cells relative to the total number of cells insix randomly selected fields of the two wells, as described (e.g., Penget al., (2010) Sci Signal., 3(122): ra38). Similar assays are performedwith primary microglial cells.

Example 21: Screening for Anti-TREM2, Anti-DAP12, and/or TEM2/DAP12Bispecific Antibodies that Induce Osteoclast Differentiation

BMM cells are seeded onto the plates in triplicate wells and treatedwith RANKL, M-CSF, and with an anti-TREM2, anti-DAP12, and/orTREM2/DAP12 bispecific antibody, or an isotype-matched controlmonoclonal antibody. The medium is changed every 3 days until largemultinucleated cells are visible. After 3 to 5 days in culture, cellsare fixed with 3.7% formaldehyde in PBS for 10 min. Plates are thenwashed twice in PBS, incubated for 30 s in a solution of 50% acetone and50% ethanol, and washed with PBS. Cells are stained fortartrate-resistant acid phosphatase (TRAP) with a kit from Sigma(product 435). Multinucleated (more than two nuclei), TRAP-positivecells are then counted by light microscopy, as described (e.g., Peng etal., (2010) Sci Signal., 3(122): ra38).

Example 22: Screening for Anti-TREM2, Anti-DAP12, and/or TEM2/DAP12Bispecific Antibodies that Normalize TREM2/TYROBP-Dependent Changes inGene Expression within the Immune/Microglia Regulatory Module

Microglial cells derived from mouse embryonic stem cells are geneticallymodified by lentiviral vectors to overexpress either full-length or atruncated version of Tyrobp that lacks both intracellular immunoreceptortyrosine-based activation motif (ITAM) motifs. Microglia cells are alsoderived from mouse embryonic stem cells that are heterozygous for TREM2.To assess the genome-wide gene-expression changes in response to theperturbation of Tyrobp or TREM2, gene-expression data is derived fromthe RNA sequencing of mouse microglial cell lines overexpressing: (1)vehicle, (2) full-length Tyrobp, or (3) dominant-negative truncatedTyrobp; or (4) overexpressing a knockdown construct for TREM2, such asSiRNA and cells which are heterozygous for TREM2. Approximately 2,638and 3,415 differentially expressed genes for the overexpression offull-length Tyrobp and truncated Tyrob are identified, respectively(Zhang et al., (2013) Cell 153, 707-720). Approximately 99% of thedifferentially expressed genes from the microglia overexpressing intactTyrobp are downregulated compared to the control vehicle. For example,658 genes, related to the vacuole/autophagy, as well as genes involvedwith RNA metabolism and cell-cycle mitosis are downregulated by activeTyrobp, but upregulated in cells expressing dominant-negative truncatedTyrobp. Conversely, some 2,856 genes for the vacuole/autophagy pathwayand for mitochondrion are selectively upregulated in microgliaexpressing the dominant-negative truncated Tyrobp.

Agonistic anti-TREM2, anti-DAP12, and/or TREM2/DAP12 bispecificantibodies are screened for their ability to elicit gene expressionprofiles similar to that observed in normal microglial cells and inmicroglial cells overexpressing intact Tyrobp in cells that expressdominant-negative truncated Tyrobp (Zhang et al., (2013) Cell 153,707-720), in cells that express the knockdown construct for TREM2, or incells that are heterozygous for TREM2. Antibodies that are capable ofchanging the gene expression network are selected.

Example 23: TREM2 Antibodies Induce the Expression of CD83 and CD86 onHuman Dendritic Cells (DCs)

To evaluate the ability of anti-TREM2 antibodies to modify expression ofCD83 and CD86, both plate bound and soluble antibodies were incubatedwith dendritic cells (DCs), and the expression of CD83 and CD86 weremeasured.

Antibodies were plated overnight at 4° C. in 12 well plates at 2 or 5ug/ml in PBS. Wells were washed 3× with PBS the next day. On day 5,immature human DCs were harvested and plated at 1 million cells per welland incubated at 37C, 5% CO₂ in the absence of cytokine. FACS analysisof CD86, CD83, CD11c, HLA-DR, and LIN (BD Biosciences) was performed ona BD FACS Canto 48 hours later. Data analysis was performed with FlowJo(TreeStar) software version 10.0.7. Levels of CD83 and CD86 wereevaluated on CD11c+HLA-DR+LIN− cell populations.

Alternatively, Day 5 immature human dendritic cells were plated at100,000 cells per well in a U-bottom non-TC treated 96 well plate inmedia without cytokine. Antibodies were added at 5 ug/ml with or withoutLPS-removed anti-human secondary (Jackson ImmunoResearch) at 20 ug/ml.FACS analysis for CD86, CD83, CD1 c, HLA-DR, and LIN (BD Biosciences)was performed 48 hrs post antibody addition as previously described.

Plate bound TREM2 antibodies Ab21 and Ab52 increased the frequency ofCD83+CD86+ DCs compared to the isotype control antibody Ab88 (FIG. 5A).Soluble antibody Ab21 and Ab52, both alone and cross-linked with ananti-human secondary antibody, induced equivalent expression of CD86 onDCs to the isotype control antibody (Ab88) (FIG. 5B). Based on theseresults, TREM2 antibodies Ab21 and Ab52 function as agonists to inducethe expression of inflammatory surface markers CD83 and CD86 on humandendritic cells.

Example 24: TREM2 Antibodies Ab21 and Ab52 Induce Syk Phosphorylation

Spleen tyrosine kinase (Syk) is an intracellular signaling molecule thatfunctions downstream of TREM2 by phosphorylating several substrates,thereby facilitating the formation of a signaling complex leading tocellular activation and inflammatory processes. The ability of agonistTREM2 antibodies to induce Syk activation was determined by culturinghuman and mouse macrophages and primary human dendritic cells andmeasuring the phosphorylation state of Syk protein in cell extracts.

Bone marrow-derived macrophages (BMDM), WT mouse BMDM, TREM2 knockout(KO) mouse BMDM, and primary human dendritic cells were starved for 4hours in 1% serum RPMI and then removed from tissue culture dishes withPBS-EDTA, washed with PBS, and counted. The cells were coated with thefull-length agonist TREM2 antibodies Ab21 and Ab52, or controlantibodies (Ab89 or Ab92) for 15 minutes on ice. After washing with coldPBS, cells were incubated at 37° C. for the indicated period of time inthe presence of goat anti-human IgG. After stimulation, cells were lysedwith lysis buffer (1% v/v NP-40%, 50 Mm Tris-HCl (pH 8.0), 150 mM NaCl,1 mM EDTA, 1.5 mM MgCl2, 10% glycerol, plus protease and phosphataseinhibitors) followed by centrifugation at 16,000 g for 10 min at 4° C.to remove insoluble materials. Lysates were then immunoprecipitated withanti-Syk Ab (N-19 for BMDM or 4D10 for human DCs, Santa CruzBiotechnology). Precipitated proteins were fractionated by SDS-PAGE,transferred to PVDF membranes and probed with anti-phosphotyrosine Ab(4G10, Millipore). To confirm that all substrates were adequatelyimmunoprecipitated, immunoblots were reprobed with anti-Syk Ab (Abcam,for BMDM) or anti-Syk (Novus Biological, for human DCs). Visualizationwas performed with the enhanced chemiluminescence (ECL) system (GEhealthcare), as described (e.g., Peng et al., (2010) Sci Signal.,3(122): ra38).

TREM2 antibodies Ab21 and Ab51 induced Syk phosphorylation in WT TREM2mouse BMDMs, but did not induce phosphorylation in TREM2 KO(TREM2^(−/−)) cells (FIG. 6A). Antibodies Ab21 and Ab51 also induced Sykphosphorylation in both human BMDM (FIG. 6A, right panel) and in primaryhuman dendritic cells (FIG. 6B). Control antibodies Ab89 and Ab92 didnot induce Syk phosphorylation. Based on these results, TREM2 antibodiesAb21 and Ab52 function as agonists to induce Syk phosphorylation inmacrophages and dendritic cells.

Example 25: TREM2 Antibodies Ab21 and Ab52 Induce DAP12 Phosphorylationin Mouse Macrophages

TREM2 signals through DAP12, leading downstream to activation of PI3Kand other intracellular signals. The ability of agonist TREM2 antibodiesto induce DAP12 activation was determined by culturing mouse macrophagesand measuring the phosphorylation state of DAP12 protein in cellextracts.

Before stimulation with antibodies, mouse wild-type (WT) bonemarrow-derived macrophages (BMDM) and TREM2 knockout (KO) BMDM werestarved for 4h in 1% serum RPMI. 15×10⁶ cells were incubated in ice for15 min with full-length agonistic or control antibodies. Cells werewashed and incubated at 37° C. for the indicated period of time in thepresence of goat anti-human IgG. After stimulation, cells were lysedwith lysis buffer (1% v/v NP-40%, 50 Mm Tris-HCl (pH 8.0), 150 mM NaCl,1 mM EDTA, 1.5 mM MgCl₂, 10% glycerol, plus protease and phosphataseinhibitors), followed by centrifugation at 16,000 g for 10 min at 4° C.to remove insoluble materials. Cell lysate was immunoprecipitated with asecond TREM2 antibody (R&D Systems). Precipitated proteins werefractionated by SDS-PAGE, transferred to PVDF membranes, and probed withanti-phosphotyrosine Ab (4G10, Millipore). The membrane was stripped andreprobed with anti-DAP12 antibody (Cells Signaling, D7G1X). Each celllysate used for TREM2 immunoprecipitations contained an equal amount ofproteins, as indicated by a control Ab (anti-actin, Santa Cruz).

DAP12 co-precipitated with TREM2 and was phosphorylated in WTmacrophages incubated with agonist TREM2 antibodies Ab52 (FIG. 7A) andAb21 (FIG. 7B). Conversely, no DAP12 phosphorylation was observed inTREM2 KO (TREM2^(−/−)) macrophages incubated with antibody Ab21 (FIG.7B). These results demonstrate that DAP12 is associated with TREM2protein, and that agonist TREM2 antibodies are able to induce DAP12phosphorylation in vitro.

Example 26: TREM2 Antibodies Ab21 and Ab52 Compete with TREM2 Ligand forBinding to Human and Mouse TREM2

The ability of agonist TREM2 antibodies to recognize the ligand-bindingsite on TREM2 was evaluated through competitive binding assays with E.coli cells expressing a putative TREM2 ligand.

E. coli were grown in 10 ml LB media O/N, harvested by centrifuging, andwashed twice in 10 ml PBS. E. coli were then heat-inactivated byincubating in a 70° C. water bath for 30 min. E. coli were labeled withCellTracker DeepRed (ThermoFisher/Invitrogen, 1 uM final concentration)and subsequently washed thrice in 10 ml PBS and resuspended in 1 ml PBSat a concentration of 10⁸/ml. For competition binding, bacteria wereadded to a mouse TREM2 and DAP12 expressing cell line (BWZ), or to a BWcell line expressing a human TREM2/DAP12 fusion protein, together with10 μg/ml of full-length agonist TREM2 antibodies and incubated for onehour on ice. Cells were analyzed via FACS for binding of CellTrackerlabeled bacteria to the cell lines.

TREM2 antibodies Ab21 and Ab52 inhibited the binding of E. coli bacteriato both human and mouse cells, indicating competitive binding of theantibodies to the ligand-binding site on TREM2 (FIG. 8). Non-agonisticcontrol TREM2 antibodies (Ab66 and Ab68) inhibited bacterial binding tohuman cells expressing TREM2, but did not inhibit binding to mouseTREM2.

Example 27: Summary of TREM2 Antibody Functional Studies

Table 6 summarizes results of the functional studies described inExamples 24-26 above. Antibodies Ab21 and Ab52 demonstrated induction ofSyk phosphorylation in human dendritic cells (hDC), mouse dendriticcells (mDC), and mouse macrophages (mMac). However, antibody Ab52induced higher levels of phosphorylated Syk compared to antibody Ab21 inhuman and mouse DCs. Both antibodies were able to mimic ligand bindingto human and mouse TREM2, although antibody Ab21 demonstrated moreeffective binding, as indicated by a greater decrease in bacterialbinding (see Example 26 above).

TABLE 6 TREM2 Antibody Functional Studies Induction Induction InduceMimic ligand Mimic ligand Phospho Syk Phospho Phospho binding site onbinding site Antibody hDC Syk mDC DAP12 mMac human Trem2 on mouse Trem2Ab52 +++ +++ +++ ++ +++ Ab21 ++ ++ +++ +++ +++ Isotype − − − − − Control

Example 28: TREM2 Decreases the Secretion of Inflammatory Cytokines fromMouse Macrophages

In order to determine the role of TREM2 in inflammatory cytokineproduction, mouse wild-type (WT), TREM2 knock-out (KO), and TREM2heterozygous (Het) macrophages were cultured with various inflammatorymediators, and cytokine levels were measured in the culturesupernatants.

To generate BMDM, total bone marrow from wild-type (WT), TREM2 KO (KO),and TREM2 heterozygous (Het) mice was cultured in RPMI supplemented with10% bovine calf serum, 5% horse serum, and 50 ng/ml recombinant mouseCSF-1 (R&D Systems). Cells were cultured for 5 days, and adherent cellswere detached with 1 mM EDTA in PBS. BMDM were plated on 96-well platesat 105 cells/well and allowed to adhere for 4 h at 37° C. Cells werethen stimulated with TLR agonists LPS (Salmonella abortus equi) orzymosan (Saccharomyces cerevisiae) at concentrations ranging from0.01-100 ng/ml (LPS) or 0.01-100 μg/ml (zymosan). Alternatively,macrophages isolated from WT, KO, and Het mice were cultured in thepresence of 10 ng/ml of the cytokine IL-4 or 50 ng/ml of IFN-γ. Cellculture supernatant was collected 24 or 48 h after stimulation and thelevels of TNFa, IL-6, IL-10, and MCP-1 cytokines were measured by usingCytometric Bead Array Mouse Inflammation Kit (BD) according tomanufacturer's protocol.

Wild-type (WT) macrophages stimulated with the inflammatory mediatorsLPS or zymosan secreted less inflammatory cytokines TNFa, IL-6, IL-10,and MCP-1 compared to TREM2 KO (TREM2^(−/−)) macrophages (FIG. 9A).Similarly, WT and Het (TREM2^(+/−)) macrophages treated with themediator IFNγ produced less inflammatory cytokines IL-6 and TNFacompared to TREM2 KO macrophages (FIG. 9B). WT, Het, and KO macrophagescultured in the presence of the cytokine IL-4 produced similar lowlevels of IL-6 and TNFa (FIG. 9B). Based on these results, TREM2antibodies may reduce the secretion of inflammatory cytokines frommacrophages.

Example 29: TREM2 Decreases the Expression of Inflammatory Cell SurfaceMarkers on Mouse Macrophages

In order to determine the role of TREM2 in inflammatory markerexpression, mouse wild-type (WT), TREM2 knock-out (KO), and TREM2heterozygous (Het) macrophages were cultured with various inflammatorymediators, and the expression of surface markers CD86 and CD206 weremeasured.

Macrophages isolated from WT, KO, and Het mice were plated and allowedto adhere for 4 h at 37° C., and TLR agonists LPS (Salmonella abortusequi) and zymosan (Saccharomyces cerevisiae) were added atconcentrations ranging from 0.01-100 ng/ml (LPS) or 0.01-10 μg/ml(zymosan). Alternatively, macrophages isolated from WT, KO, and Het micewere cultured in the presence of the cytokines IL-4 (10 ng/ml) or IFN-γ(0.5-50 ng/ml). FACS analysis of CD86 and CD206 was performed on a BDFACS Canto 48 hours later. Data analysis was performed with FlowJo(TreeStar) software version 10.0.7.

Wild-type (WT) macrophages treated with the inflammatory mediators IFN-γ(FIG. 10A), LPS, or Zymosan (FIG. 10B) expressed lower levels of theinflammatory receptor CD86 but not of the receptor CD206 compared TREM2KO (TREM2^(−/−)) macrophages. Similarly, Het (TREM2^(+/−)) macrophagestreated with I IFN-γ expressed lower levels of CD86 but not of CD206compared to TREM2 KO macrophages (FIG. 10A). Based on these results,TREM2 agonistic antibodies may reduce expression of inflammatoryreceptors on macrophages.

Example 30: TREM2 Increases the Survival of Macrophages and DendriticCells

To evaluate the role of TREM2 in cell survival, wild-type (WT) and TREM2knock-out (KO) macrophages and dendritic cells were cultured in thepresence of inflammatory mediators, and cell survival was measured.

Murine bone marrow precursor cells from TREM2 WT, Het, and KO mice wereobtained by flushing tibial and femoral marrow cells with cold PBS.After one wash with PBS, erythrocytes were lysed using ACK Lysing Buffer(Lonza), washed twice with PBS and suspended at 0.5×10⁶ cells/ml incomplete RPMI media (10% FCS, Pen/Strep, Gln, neAA) with the indicatedamounts of 50 ng/ml M-CSF to produce macrophages, or 10 ng/ml GM-CSF toproduce dendritic cells. For M2-type macrophages, 10 ng/ml IL-4 wasadded to the cultured cells. For M1-type macrophages, 50 ng/ml IFN-γ wasadded. In some experiments LPS or zymosan was added to the cell cultureat day 5 at a concentration range of 1 μg/ml-0.01 ng/ml. Recombinantcytokines were purchased from Peprotech.

To analyze viability of bone marrow-derived macrophages, cells wereprepared as above and cultured in MCSF. Cells were either plated at10⁵/200 μl in a 96-well plate (for viability analysis using a luciferasebased-assay) or at 0.5×10⁶/1 ml in a 6-well plate (for Tripan Blueexclusion cell count) in non-tissue culture treated plates. Mediacontaining fresh M-CSF was added at day 3. At the indicated time pointscells were gently detached from the plates with 3 mM EDTA and countedusing a Burker chamber. For FACS analysis of live cells, macrophageswere cultured either in 50 ng/ml MCSF for 6 days (+MCSF) or in 50 ng/mlMCSF for 4 days before MCSF was removed for an additional 36 hrs(−MCSF). Cells were stained using CD11b antibody and DAPI. Forluciferase viability assays, cell viability was measured at day 5 ofculture in graded concentrations of growth factors GMCSF (dendriticcells), MCSF (M1 macrophages), or MCSF+IL-4 (M2 macrophages). Cells weredirectly incubated with ToxGlo reagent (Promcga) and luciferase activity(luminescence) was read using an XY reader. For FACS analysis of viablemacrophages cultured in the presence of inflammatory mediators IFN-γ,LPS, or zymosan, cells were collected at day 5 and stained using CD11bantibody and DAPI.

After 7 days of culture in MCSF, significantly higher numbers of viable(trypan blue excluded) TREM2 WT and Het (TREM2^(+/−)) macrophages wereobserved than TREM2 KO (TREM2^(−/−)) macrophages (FIG. 11A). FACSanalysis revealed that WT macrophages treated with MCSF for either 6(+MCSF) or 4 days (−MCSF) displayed increased survival compared to Hetand KO macrophages, as indicated by a higher percentage of live(CD11b+DAPI−) cells (FIG. 11B). For luciferase assays, WT cells culturedin the presence of growth factors GMCSF (dendritic cells), MCSF (M1macrophages), or MCSF+IL-4 (M2 macrophages) survived better than KOcells, as indicated by a higher luminescence reading across the range ofgrowth factor concentrations (FIG. 11C). Wild-type macrophages culturedwith inflammatory mediators (IFN-γ, LPS, or zymosan) had a highersurvival rate than TREM2 Het and KO macrophages, as indicated by ahigher percentage of CD11b+ live cells (FIG. 11D). Based on theseresults, TREM2 agonistic antibodies may increase the survival ofmacrophages and dendritic cells, while TREM2 antagonistic antibodies candecrease cell survival.

Example 31: TREM2 Modulates Phagoctosis

TREM2 signaling is involved in phagocytosis pathways, includingbacterial clearance from the lung and phagocytosis of apoptotic neurons.The role of TREM2 in phagocytosis was evaluated by measuring the abilityof wild-type (WT) and TREM2 knock-out (KO) mouse macrophages tophagocytose E. coli cells and apoptotic cells.

WT and TREM2 KO BMDM were starved in 1% serum RPMI or kept in culture inthe presence of MCSF (50 ng/ml) overnight. The following day cells wereplated at 2×10⁵ in 96 well plates (round bottom non tissue culture), inthe presence or absence of MCSF. Target cells (CCL119) were culturedovernight with 0.5 μM staurosporine to induce apoptosis. The day aftercells were washed and labeled with 20 ng/ml of pHrodo-SE (Invitrogen).Apoptotic cells or bioparticle E. coli (pHrodo, Invitrogen) were addedand the cells were incubated at 37° C. for 1 h and 30 min. The assay wasstopped on ice. After washing with cold PBS, cells were stained forCD11b (pacific blue-CD11b, BD) and analyzed by FACS. In all samples,BMDM were distinguished from apoptotic cells and beads by CD11bstaining, and gates were drawn based on effector cells cultured withouttarget cells. For negative controls, effector cells were incubatedduring the entire assay with cytochalasin D (2 μM, SIGMA). Phagocytosiswas quantified as (percent or MFI of PhRodo positive cells)−(percent orMFI of PhRodo positive cytochalasin D treated negative control cells).

Wild-type (WT) macrophages cultured with MCSF displayed lessphagocytosis of apoptotic cells and E. coli cells as compared to TREM2KO (TREM2^(−/−)) macrophages, as indicated by a lower percentage ofpHrodo+ cells (FIG. 12). Conversely, WT macrophages cultured withoutMCSF displayed increased phagocytosis of apoptotic cells and E. colicells as compared to TREM2 KO macrophages, as indicated by a higherpercentage of pHrodo+ cells (FIG. 12). Based on these results, TREM2agonistic antibodies may enhance phagocytosis in the absence of MCSF andreduce phagocytosis in the presence of MCSF. Conversely, it is believedthat TREM2 antagonistic antibodies can enhance phagocytosis in thepresence of MCSF and reduce phagocytosis in the absence of MCSF.

Example: 32: Epitope Mapping of TREM2 Antibodies

TREM2 antibodies were tested for their ability to bind 15 or 25 merpeptides spanning the entire human and mouse TREM2.

Linear 15-mer peptides were synthesized based on the sequence of humanor mouse TREM2, with a 14 residue overlap. In addition, linear 25-merpeptides were synthesized based on sequence of human or mouse TREM2 witha 1 residue shift. The binding of TREM2 antibodies to each of thesynthesized peptides was tested in an ELISA-based method. In this assay,the peptide arrays were incubated with primary antibody solution(overnight at 4° C.). After washing, the peptide arrays were incubatedwith a 1/1000 dilution of an antibody peroxidase conjugate (SBA, cat.nr. 2010-05) for one hour at 25° C. After washing, the peroxidasesubstrate 2,2′-azino-di-3-ethylbenzthiazoline sulfonate (ABTS) and 2μl/ml of 3% H2O2 were added. After one hour, the color development wasmeasured. The color development was quantified with a charge coupleddevice (CCD) camera and an image processing system.

Antibody Ab52 demonstrated reliable binding in all peptide sets.Antibody Ab52 was found to recognize an N-terminal peptide regionbetween amino acid residues 49-57 of human and mouse TREM2(⁴⁹AWCRQLGEK⁵⁷ (SEQ ID NO: 444)) (FIG. 13). The epitope regionrecognized by Ab52 corresponds to amino acid residues 49-57 of SEQ IDNO: 1 and has the amino acid sequence of: AWCRQLGEK (SEQ ID NO: 444).

Antibody Ab21 was found to recognize an N-terminal peptide regionbetween amino acid residues 43-50 of human and mouse TREM2 (⁴³HWGRRAW⁵⁰(SEQ ID NO: 445)). The epitope region recognized by Ab21 corresponds toamino acid residues 43-50 of SEQ ID NO: 1 and has the amino acidsequence of: HWGRRAW (SEQ ID NO: 445).

Example 33: Comparison of Agonistic TREM2 Antibodies Ab52 and Ab21 toReference TREM2 Antibodies

Reference TREM2 antibodies were compared to agonist antibodies Ab21 andAb52 by evaluating their ability to induce Syk phosphorylation and bydetermining their TREM2 binding region.

Cells were coated with 1 μg/10⁶ cells of TREM2 antibody MAB17291 (R&DSystems) or monoclonal rat IgG1 antibody 78.18 (obtained from theUniversity of California, San Francisco) and stimulated by cross-linkingwith a secondary antibody (goat anti-rat 1.5 μg/10⁶ cells). Sykphosphorylation was evaluated according to the methods described inExample 24 above.

To evaluate antibody binding regions, human TREM2-Fc was incubated withimmobilized full-length agonist TREM2 antibodies Ab21 or Ab52, and TREM2antibodies MAB17291 or 78.18 were subsequently added. Epitope binning ofthe antibodies was performed on a Forte Bio Octet Red384 system (PallForte Bio Corporation, Menlo Park, Calif.) using a standard sandwichformat binning assay (see Estep et al, (2013) MAbs 5(2):270-8). Controlanti-target IgG was loaded onto AHQ sensors and unoccupied Fc-bindingsites on the sensor were blocked with a non-relevant human IgG1antibody. The sensors were then exposed to 100 nM target antigenfollowed by a second anti-target antibody. Data was processed usingForteBio's Data Analysis Software 7.0. Additional binding by the secondantibody after antigen association indicates an unoccupied epitope(non-competitor), while no binding indicates epitope blocking(competitor).

Reference TREM2 antibody MAB17291 induced Syk phosphorylation in WT, butnot TREM2 KO (TREM2^(−/−)) cells. Reference TREM2 antibody 78.18,however, did not induce Syk phosphorylation under the experimentalconditions used (FIG. 14). This is in contrast to Ab21 and Ab52, whichwere both able to induced Syk phosphorylation in WT TREM2 mouse BMDMs(FIG. 6).

Reference antibody MAB17291 was able to simultaneously bind TREM2 withantibody Ab21 (FIG. 15A) or antibody Ab52 (FIG. 15B). These resultsdemonstrate that agonist TREM2 antibodies Ab21 and Ab52 bind differentregions of the TREM2 protein than does reference antibody MAB17291.

Example 34: Analysis of Ability of TREM2 Antibody Fabs to StimulateViability of Innate Immune Cells

The agonistic functionality of plate bound, cross-linked anti-TREM2antibody Fab fragments derived from antibodies Ab21 and Ab52 wasevaluated in innate immune cells (e.g., macrophages).

Wild-type (WT) and TREM2 knock-out (KO) mouse bone marrow derivedmacrophages were cultured in the presence of M-CSF and plate bound TREM2antibody Fabs, and cell viability was measured.

Macrophages isolated from the bone marrow of WT and KO mice were platedon non-tissue-culture-treated 96-well plates, pre-coated with either12.5 nM or 100 nM of cross-linked Ab21 or Ab52 Fabs. Cells were culturedfor 48 hours in the presence of 10 ng/ml M-CSF. Analysis of viabilitywas performed using Cell Titer Glo kit (Promega). Plates were read witha BioTek Synergy Microplate Reader using GEN5 2.04 software.

Cross-linked TREM2 Fab fragments derived from antibodies Ab21 and Ab52increased the number of viable mouse bone marrow-derived macrophagescompared to isotype control Fab Ab88, as indicated by a higher %increased survival (FIG. 16). This enhancement in cell viability was notobserved in KO mouse macrophages. These data indicate that thebiological activity of TREM2 antibodies Ab21 and Ab52 is TREM2 specific,and that plate bound, cross-linked Ab21 and Ab52 Fab fragments functionas agonists to increase the survival of macrophages cultured in M-CSF.

Example 35: Analysis of Ability of TREM2 Antibodies to Decrease Survivalof Innate Immune Cells

The antagonistic functionality of both soluble, non-cross-linkedanti-TREM2 antibody Fab fragments derived from antibodies Ab21 and Ab52and soluble, full-length anti-TREM2 antibodies Ab21 and Ab52 wasevaluated in innate immune cells (e.g., macrophages).

Wild-type (WT) and TREM2 knock-out (KO) mouse bone marrow derivedmacrophages were cultured in the presence of M-CSF and soluble TREM2antibody Fabs or soluble full-length antibodies, and cell viability wasmeasured.

Macrophages isolated from the bone marrow of WT and KO mice were platedon non-tissue-culture-treated 96-well plates in the presence of 20 ng/mlM-CSF and increasing amounts of the indicated soluble, non-cross-linkedTREM2 antibody Fabs or soluble, full-length antibodies. Each conditionwas plated in triplicate. Analysis of viability was performed using CellTiter Glo kit (Promega) 3 days later. Plates were read with a BioTekSynergy Microplate Reader using GEN5 2.04 software.

In FIG. 17, the “NT” dotted line indicates the average cell viabilityobtained with untreated macrophages (no antibody added). The “no MCSF”dotted line indicates the average cell viability obtained whenmacrophages were cultured in the absence of M-CSF.

When macrophage cell viability was evaluated with soluble,non-cross-linked TREM2 antibody Fabs, the results indicated thatsoluble, non-cross-linked TREM2 Fab fragments derived from antibody Ab52decreased cell viability (FIG. 17A). In contrast, soluble,non-cross-linked TREM2 Fab fragments derived from antibody Ab21 did notinhibit viability and had an effect comparable to the isotype controlAb99 (FIG. 17A). The results demonstrate that the soluble,non-cross-linked Fab derived from TREM2 antibody Ab52 can function as anantagonist and inhibit the survival of macrophages in vitro.

When macrophage cell viability was evaluated with soluble, full-lengthantibodies, antibody Ab52 decreased cell viability more potently thandid Ab21 (FIG. 17B). Indeed, soluble, full-length antibody Ab21 had aneffect on cell that was comparable to that of isotype control Ab91 (FIG.17B). The results demonstrate that soluble, full-length TREM2 antibodyAb52 can function as antagonist, when not cross-linked or clustered, andinhibit the survival of macrophages.

The results of these experiments indicate that the TREM2 antibody Ab52,in the absence of clustering, can inhibit the survival of innate immunecells such as macrophages. In contrast, the TREM2 antibody Ab21, even inthe absence of clustering, does not inhibit the survival of innateimmune cells such as macrophages.

Example 36: Analysis of the Ability of TREM2 Antibodies to InduceTREM2-Dependent Genes

The ability of plate bound anti-TREM2 antibodies Ab21 and Ab52 toactivate TREM2-dependent genes was evaluate using a luciferase reportergene under the control of an NFAT (nuclear factor of activated T-cells)promoter.

A cell line derived from mouse thymus lymphoma T lymphocytesBW5147.G.1.4 (ATCC® TIB48™) was infected with mouse Trem2 and Dap12, andwith Cignal Lenti NFAT-Luciferase virus (Qiagen). Full-length anti-TREM2antibodies were plate bound at 10 ug/ml in DPBS on tissue-culturetreated clear bottom white 96 well plates (100 ul/well), overnight at 4°C. Wells were rinsed thrice with DPBS and subsequently were plated at100,000 cells/well in media with 1% serum. As a positive control forsignaling, PMA (0.05 ug/ml) and ionomycin (0.25 uM) were added together.Cells were incubated for 6 hours and luciferase activity was measured byadding OneGlo Reagent (Promega) to each well and incubating 3 min at RTon a plate shaker. Luciferase signal was measured using a BioTek platereader.

As shown in FIG. 18A, anti-TREM2 antibodies Ab 21 and Ab52 increasedluciferase activity, indicating that the antibodies were able to induceTREM2-dependent gene transcription.

As shown in FIG. 18B, plate bound phosphatidylserine (PS) inducesTREM2-dependent gene expression. It is believed that PS is a naturalligand of TREM2. Thus, the results in FIG. 18 indicate that agonistanti-TREM2 antibodies can mimic a natural ligand of TREM2.

The ability of plate bound anti-TREM2 Fab antibodies Ab21 and Ab52 toactivate TREM2-dependent genes was evaluated using primary mousemacrophages. Bone marrow-derived macrophages (BMM) were generated withM-CSF for 5 days. BMM (10⁵/well) was seeded on 96 well plates that hadbeen previously coated with the Fab portion of the antibodies Ab21 andAb52 (50 nM). Ultrapure LPS (100 ng/ml Escherichia coli 0111:B4) wasadded after the plates were spun for 1 min at 1200 rpm. After 18 h ofstimulation with LPS, cytokines present in cell supernatants weremeasured with cytometric bead array (CBA; BD Biosciences, CBA mouseinflammation kit).

As shown in FIGS. 18C and 18D, plate bound anti-TREM2 Fab antibodies Ab21 and Ab52 increased the levels of IL-6 and MCP-1, indicating that theantibodies were able to activate TREM2-dependent genes in primary immunecells.

Example 37: Analysis of the Ability of TREM2 Antibodies to InhibitTREM2-Dependent Genes

The ability of soluble, full-length anti-TREM2 antibodies Ab21 and Ab52to inhibit TREM2-dependent genes was evaluate using a luciferasereporter gene under the control of an NFAT (nuclear factor of activatedT-cells) promoter.

A cell line derived from mouse thymus lymphoma T lymphocytesBW5147.G.1.4 (ATCC® TIB48™) was infected with mouse Trem2 and Dap12, andwith Cignal Lenti NFAT-Luciferase virus (Qiagen). Soluble, full-lengthanti-TREM2 antibodies Ab 21 or Ab52 were added at increasingconcentration to the cells. Cells were incubated for 6 hours at 37° C.and luciferase activity was measured using OneGlo Reagent (Promega).

The cells display tonic TREM2-dependent signaling due to either thepresence of an endogenous ligand or to spontaneous receptor aggregation,which leads to TREM2 signaling.

The dotted line in FIG. 19 indicates the levels of TREM2 activitywithout stimulation.

As shown in FIG. 19, soluble, full-length anti-TREM2 antibody Ab52 wasable to inhibit tonic, TREM2-dependent gene expression. Soluble,full-length anti-TREM2 antibody Ab21 was able to partially inhibittonic, TREM2-dependent gene expression; however the levels of geneexpression were nearly the same as the levels of TREM2 activity withoutstimulation (FIG. 19).

Example 38: Summary of TREM2 Antibody Agonistic and AntagonisticActivity

Table 7 summarizes results of the functional studies described inExamples 35-37 above. Antibodies Ab21 and Ab52 demonstrated agonisticactivity in activating TREM2-dependent gene expression using either aluciferase reporter gene (FIG. 18) or a beta-GAL reporter gene (Table7). As indicated in Table 7, Ab21 showed an increased level of geneinduction, compared to Ab52 when the luciferase reporter gene was used.However, Ab52 showed an increased level of gene induction, compared toAb21 when the beta-GAL reporter gene was used (Table 7). Antibodies Ab21and Ab52 also demonstrated agonistic activity in stimulating cellsurvival of innate immune cells (FIG. 16). Table 7 further summarizesresults demonstrating the antagonistic effects of soluble,non-cross-linked antibody Ab52 in inhibiting cell survival of innateimmune cells (FIG. 17). In contrast, soluble, non-cross-linked antibodyAb21 had minimal antagonistic activity in inhibiting cell survival (FIG.17).

TABLE 7 TREM2 Antibody Agonist and Antagonist Activity Luciferasebeta-GAL Survival Luciferase Agonistic Agonistic Agonistic Antagonisticantibody antibody antibody antibody activity in solution/ Antibodyactivity activity activity antagonistic Ab format Ab52 +++ +++ +++ +++Ab21 ++++ ++ +++ −/+ Isotype − − − − control

Example 39: Analysis of Anti-Alzheimer's Disease Effect of Agonist TREM2Antibodies

To evaluate the ability of agonistic anti-TREM2 antibodies to delay,prevent, or reverse the development of Alzheimer's disease (AD), 5×FADmice are used. 5×FAD mice overexpress mutant human APP (695) with theSwedish (K670N, M671L), Florida (I716V), and London (V717I) familialAlzheimer's disease (FAD) mutations, along with human PS1 harboring twoFAD mutations, M146L and L286V. Both transgenes are regulated by themouse Thy1 promoter to drive over expression on the brain andrecapitulate major features of AD. Mice treated with the agonisticanti-TREM2 antibodies or with control antibodies are tested for A betaplaque load with immunohistochemistry and by ELISA of tissue extracts.They are further tested for the number of microglia in the brain, andfor reduction in cognitive deficit using Morris Water maze, a spatiallearning and memory task, Radial Arm Water Maze, a spatial learning andmemory task, Y Maze (quantifies spontaneous alternation as a measure ofspatial cognition), novelty preference in in an open field, operantlearning to assess learning and memory, and fear conditioning(mousebiology.org website; Wang et al., (2015) Cell. pii:S0092-8674(15)00127-0).

Example 40: Production, Identification, and Characterization of AgonistAnti-TREM2 Antibodies Introduction

Antibodies that bind the extracellular domain of TREM2, particularly theextra cellular domain (amino acid residues 19-174 of SEQ ID NO: 1) aregenerated using mouse hybridoma technology, phage display technology,and yeast display technology according to the methods described inExample 1 above. Antibodies were then screened for their ability to bindcells that express TREM2 and for their ability to activate TREM2signaling and functions in cells and in a whole animal in vivo asdescribed in Examples 41-67 below.

Results

Anti-TREM2 Antibody Production

Antibodies that bind the extracellular domain of TREM2, particularlywithin the extracellular sequences located at amino residues 113-174 ofSEQ ID NO: 1, were generated using the procedure described in Example 1.

A total of 87 antibodies were generated. The antibodies were thenscreened for TREM2 binding. Antibodies that were positive for binding toprimary cells were tested for agonistic activity. From the 87antibodies, certain antibodies (e.g., Ab1, Ab9, Ab14, Ab22, Ab45, andAb65) were selected for further analysis.

Antibody Heavy Chain and Light Chain Variable Domain Sequences

Using standard techniques, the amino acid sequences encoding the heavychain variable (FIG. 20A) and the light chain variable (FIG. 20B)domains of the generated antibodies were determined. The Kabat CDRsequences of the antibodies are set forth in Table 8.

TABLE 8 Kabat CDR sequences Antibody Name CDR H1 CDR H2 CDR H3 Ab1 FTFSSYAMS VISGSGGSTYYADS AKGTPTLLFQH VKG (SEQ ID NO: 3) (SEQ ID NO: 25)(SEQ ID NO: 50) Ab2  FTFSSSAMS AISGSGGSTYYADS AKVPSYDYWSGYSN VKG YYYYMDV(SEQ ID NO: 4) (SEQ ID NO: 26) (SEQ ID NO: 51) Ab3  GTFSSYAISGIIPIFGTANYAQKF AREQYHVGMDV QG (SEQ ID NO: 5) (SEQ ID NO: 27) (SEQ IDNO: 52) Ab4  GTFSSYAIS GIIPIFGTASYAQKFQG ARGVDSIMDY (SEQ ID NO: 5) (SEQID NO: 28) (SEQ ID NO: 53) Ab5  YTFTSYYIH IINPSGGSTSYAQKF ARAPQESPYVFDIQG (SEQ ID NO: 6) (SEQ ID NO: 29) (SEQ ID NO: 54) Ab6  YTFTSYYMHIINPGGGSTSYAQKF ARGSPTYGYLYDP QG (SEQ ID NO: 7) (SEQ ID NO: 30) (SEQ IDNO: 55) Ab7  YTFTSYYMH IINPSGGSTTYAQKF ARTSSKERDY QG (SEQ ID NO: 7) (SEQID NO: 31) (SEQ ID NO: 56) Ab8  GSISSSSYYWG SISYSGSTYYNPSLKSARGPYRLLLGMDV (SEQ ID NO: 8) (SEQ ID NO: 32) (SEQ ID NO: 57) Ab9 YSFTSYWIG IIYPGDSDTTYSPSFQG ARLHISGEVNWFDP (SEQ ID NO: 9) (SEQ ID NO:33) (SEQ ID NO: 58) Ab10 YSFTSNWIG IIYPGDSDTRYSPSF AREAGYDYGELAFDI QG(SEQ ID NO: 10) (SEQ ID NO: 34) (SEQ ID NO: 59) Ab11 YSFTTYWIGIIYPGDSDTRYSPSF ARAGHYDGGHLGM QG DV (SEQ ID NO: 11) (SEQ ID NO: 34) (SEQID NO: 60) Ab12 YSFTSYWIG IIYPGDSDTRYSPSF ARLGHYSGTVSSYG QG MDV (SEQ IDNO: 9) (SEQ ID NO: 34) (SEQ ID NO: 61) Ab13 YTFTSYGIS WISAYNGNTNYAQARGPSHYYDLA KLQG (SEQ ID NO: 12) (SEQ ID NO: 35) (SEQ ID NO: 62) Ab14GSISSGGYYWS NIYYSGSTVYNPSLKS ARGLYGYGVLDV (SEQ ID NO: 13) (SEQ ID NO:36) (SEQ ID NO: 63) Ab15 GSISSGGYYWS NIYYSGSTVYNPSLKS ARGLYGYGVLDV (SEQID NO: 13) (SEQ ID NO: 36) (SEQ ID NO: 63) Ab16 GSISSNSYYWGSIYYSGSTYYNPSLK ARGVLGYGVFDY (SEQ ID NO: 14) S (SEQ ID NO: 37) (SEQ IDNO: 64) Ab17 GSISSNSYYWG SIYYSGSTYYNPSLKS ARGVLGYGVFDY (SEQ ID NO: 14)(SEQ ID NO: 37) (SEQ ID NO: 64) Ab18 GSISSYYWS SIYYSGSTNYNPSLKSARDGGGEYPSGTPF DI (SEQ ID NO: 15) (SEQ ID NO: 38) (SEQ ID NO: 65) Ab19GSISSYYWS SIYYSGSTNYNPSLKS ARDGGGEYPSGTPF DI (SEQ ID NO: 15) (SEQ ID NO:38) (SEQ ID NO: 65) Ab20 GSISSYYWS SIYYSGSTNYNPSLKS ARSGMASFFDY (SEQ IDNO: 15) (SEQ ID NO: 38) (SEQ ID NO: 66) Ab22 YSFTTYWIG IIYPGDSDTRYSPSFARAGHYDGGHLGM QG DV (SEQ ID NO: 11) (SEQ ID NO: 34) (SEQ ID NO: 60) Ab23FTFSSYAMS AISGSGGSTYYADS AKLGGHSMDV VKG (SEQ ID NO: 3) (SEQ ID NO: 26)(SEQ ID NO: 67) Ab24 FTFSSYAMS AISGSGGSTYYADS AKPLKRGRGFY VKG (SEQ IDNO: 3) (SEQ ID NO: 26) (SEQ ID NO: 68) Ab25 FTFSSYAMS VISGSGGSTYYADSAKEGRTITMD VKG (SEQ ID NO: 3) (SEQ ID NO: 25) (SEQ ID NO: 69) Ab26FTFSSYAMS VISGSGGSTYYADS AKDQYSVLDY VKG (SEQ ID NO: 3) (SEQ ID NO: 25)(SEQ ID NO: 70) Ab27 FTFSSYAMS AISGSGGSTYYADS AKKYSSRGVYFDY VKG (SEQ IDNO: 3) (SEQ ID NO: 26) (SEQ ID NO: 71) Ab28 FTFSSYAMS AISGSGGSTYYADSARLGGAVGARHVTY VKG FDY (SEQ ID NO: 3) (SEQ ID NO: 26) (SEQ ID NO: 72)Ab29 FTFSSYGMH VISYDGSNKYYADS ARGQYYGGSGWFDP VKG (SEQ ID NO: 16) (SEQ IDNO: 39) (SEQ ID NO: 73) Ab30 FTFSSYAMS AISGSGGSTYYADS ARLGQEYAYFQH VKG(SEQ ID NO: 3) (SEQ ID NO: 26) (SEQ ID NO: 74) Ab31 FTFSSYGMHLIWYDGSNKYYADS ARRRDGYYDEVFDI VKG (SEQ ID NO: 16) (SEQ ID NO: 40) (SEQID NO: 75) Ab32 FTFSSYAMS AISGSGGSTYYADS ARVPKHYVVLDY VKG (SEQ ID NO: 3)(SEQ ID NO: 26) (SEQ ID NO: 76) Ab33 FTFSSYGMH VISYDGSNKYYADS ARAGGHLFDYVKG (SEQ ID NO: 16) (SEQ ID NO: 39) (SEQ ID NO: 77) Ab34 FTFSSYGMHVISYDGSNKYYADS ARDRGGEYVDFAFDI VKG (SEQ ID NO: 16) (SEQ ID NO: 39) (SEQID NO: 78) Ab35 FTFSSYAMS AISGSGGSTYYADS ARTRSGYGASNYFDY VKG (SEQ ID NO:3) (SEQ ID NO: 26) (SEQ ID NO: 79) Ab36 FTFSTYGMH VIWYDGSNKYYADSARGTGAAAASPAFDI VKG (SEQ ID NO: 17) (SEQ ID NO: 41) (SEQ ID NO: 80) Ab37FTFSSYAMS AISGSGGSTYYADS ARVGQYMLGMDV VKG (SEQ ID NO: 3) (SEQ ID NO: 26)(SEQ ID NO: 81) Ab38 FTFSTYGMH VIWYDGSNKYYADS ARGAPVDYGGIEPE VKG YFQH(SEQ ID NO: 17) (SEQ ID NO: 41) (SEQ ID NO: 82) Ab39 FTFSSYAMSAISGSGGSTYYADS AKHYHVGIAFDI VKG (SEQ ID NO: 3) (SEQ ID NO: 26) (SEQ IDNO: 83) Ab40 FTFSSYAMS AISGSGGSTYYADS ARTRSGYGASNYFDY VKG (SEQ ID NO: 3)(SEQ ID NO: 26) (SEQ ID NO: 79) Ab41 FTFSTYAMS AISGSGGSTYYADSARAMARKSVAFDI VKG (SEQ ID NO: 18) (SEQ ID NO: 26) (SEQ ID NO: 84) Ab42FTFSSSAMS AISGSGGSTYYADS AKVPSYQRGTAFDP VKG (SEQ ID NO: 4) (SEQ ID NO:26) (SEQ ID NO: 85) Ab43 FTFSSSAMS AISGSGGSTYYADS AKSPAVAGIYRADY VKG(SEQ ID NO: 4) (SEQ ID NO: 26) (SEQ ID NO: 86) Ab44 FTFSTYGMHVIWYDGSNKYYADS ARGTGAAAASPAFDI VKG (SEQ ID NO: 17) (SEQ ID NO: 41) (SEQID NO: 80) Ab45 YTFTSYYMH IINPSGGSTSYAQKF ARGPGYTTALDYYY QG MDV (SEQ IDNO: 7) (SEQ ID NO: 29) (SEQ ID NO: 87) Ab46 YTFTSYYMH IINPSGGSTSYAQKFARPAKTADY QG (SEQ ID NO: 7) (SEQ ID NO: 29) (SEQ ID NO: 88) Ab47YTFTSYYMH IINPSGGSTTYAQKF ARPGKSMDV QG (SEQ ID NO: 7) (SEQ ID NO: 31)(SEQ ID NO: 89) Ab48 YTFTSYYMH IINPSGGSTTYAQKF ARPGKSMDV QG (SEQ ID NO:7) (SEQ ID NO: 31) (SEQ ID NO: 89) Ab49 YTFTSYYMH IINPSGGSTSYAQKFARPAKTADY QG (SEQ ID NO: 7) (SEQ ID NO: 29) (SEQ ID NO: 88) Ab50YTFTSYYIH IINPSGGSTSYAQKF ARAPQESPYVFDI QG (SEQ ID NO: 6) (SEQ ID NO:29) (SEQ ID NO: 54) Ab51 YTFTSYYMH IINPSGGSTSYAQKF ARGVGGQDYYYMDV QG(SEQ ID NO: 7) (SEQ ID NO: 29) (SEQ ID NO: 90) Ab53 YTFTSYYIHIINPSGGSTSYAQKF ARAPQESPYVFDI QG (SEQ ID NO: 6) (SEQ ID NO: 29) (SEQ IDNO: 54) Ab54 YTFTSYYMH IINPSGGSTSYAQKF ARGPGYTTALDYYY QG MDV (SEQ ID NO:7) (SEQ ID NO: 29) (SEQ ID NO: 87) Ab55 YTFTGSYMH WINPNSGGTNYAQKARGPLYHPMIFDY FQG (SEQ ID NO: 19) (SEQ ID NO: 42) (SEQ ID NO: 91) Ab56YTFTGYYMH SINPNSGGTNYAQK ARASSVDN FQG (SEQ ID NO: 20) (SEQ ID NO: 43)(SEQ ID NO: 92) Ab57 YTFTNYGIS WISAYNGNTNYAQ ARGPTKAYYGSGSY KLQG VVFDP(SEQ ID NO: 21) (SEQ ID NO: 35) (SEQ ID NO: 93) Ab58 YSFTSYWIGIIYPGDSDTRYSPSF ARLGIYSTGATAFDI QG (SEQ ID NO: 9) (SEQ ID NO: 34) (SEQID NO: 94) Ab59 YTFTGSYMH WINPNSGGTNYAQK ARGGVWYSLFDI FQG (SEQ ID NO:19) (SEQ ID NO: 42) (SEQ ID NO: 95) Ab60 YTFTGYYMH WINPNSGGTSYAQKARASKMGDD FQG (SEQ ID NO: 20) (SEQ ID NO: 44) (SEQ ID NO: 96) Ab61YTFTSYGIH WISAYNGNTNYAQ ARGGVPRVSYFQH KLQG (SEQ ID NO: 22) (SEQ ID NO:35) (SEQ ID NO: 97) Ab62 YSFTSYWIG IIYPGDSDTRYSPSF ARAGHYDDWSGLG QG LDV(SEQ ID NO: 9) (SEQ ID NO: 34) (SEQ ID NO: 98) Ab63 YTFTSYGISWISTYNGNTNYAQK ARGSGSGYDSWYD LQG (SEQ ID NO: 12) (SEQ ID NO: 45) (SEQ IDNO: 99) Ab64 YSFTSYWIG IIYPGDSDTRYSPSF ARLGRWSSGSTAFDI QG (SEQ ID NO: 9)(SEQ ID NO: 34) (SEQ ID NO: 100) Ab65 YSFTSYWIG IIYPGDSDTRYSPSFARLGRKPSGSVAFDI QG (SEQ ID NO: 9) (SEQ ID NO: 34) (SEQ ID NO: 101) Ab66YTFTGSYMH WINPNSGGTNYAQK ARAGHKTHDY FQG (SEQ ID NO: 19) (SEQ ID NO: 42)(SEQ ID NO: 102) Ab67 YTFTSYYMH IINPSGGSTTYAQKF ARPGKSMDV QG (SEQ ID NO:7) (SEQ ID NO: 31) (SEQ ID NO: 89) Ab68 FTFSSYGMH LIWYDGSNKYYADSAKPGSMTDY VKG (SEQ ID NO: 16) (SEQ ID NO: 40) (SEQ ID NO: 103) Ab69YTFTGSYMH WINPNSGGTNYAQK ARAKSVDHDY FQG (SEQ ID NO: 19) (SEQ ID NO: 42)(SEQ ID NO: 104) Ab70 YTFTGYYMH WINPNSGGTSYAQK ARASKMGDD FQG (SEQ ID NO:20) (SEQ ID NO: 44) (SEQ ID NO: 96) Ab71 YTFTSYYMH IINPSGGSTSYAQKFARDISTHDYDLAFDI QG (SEQ ID NO: 7) (SEQ ID NO: 29) (SEQ ID NO: 105) Ab72GSISSYYWS SIYYSGSTNYNPSLKS ARSGTETLFDY (SEQ ID NO: 15) (SEQ ID NO: 38)(SEQ ID NO: 106) Ab73 YSFTSYWIG IIYPGDSDTTYSPSFQG ARAKMLDDGYAFDI (SEQ IDNO: 9) (SEQ ID NO: 33) (SEQ ID NO: 107) Ab74 YTFTGSYMH WINPNSGGTNYAQKARAGHKTHDY FQG (SEQ ID NO: 19) (SEQ ID NO: 42) (SEQ ID NO: 102) Ab75YTFTGYYMH WINPNSGGTNYAQK ARDLGYSSLLALDI FQG (SEQ ID NO: 20) (SEQ ID NO:42) (SEQ ID NO: 108) Ab76 FTFSSYSMN SISSSSSYIYYADSVKG ARGGGRRGDNNWF DP(SEQ ID NO: 23) (SEQ ID NO: 46) (SEQ ID NO: 109) Ab77 FTFSSYGMHVISYDGSNKYYADS ARGPPHEMDY VKG (SEQ ID NO: 16) (SEQ ID NO: 39) (SEQ IDNO: 110) Ab78 FTFSSYGMH VIWYDGSNKYYADS ARTPYPWIYFDL VKG (SEQ ID NO: 16)(SEQ ID NO: 41) (SEQ ID NO: 111) Ab79 FTFSSYSMN YISGSSSTIYYADSVARGGRRHYGGMDV KG (SEQ ID NO: 23) (SEQ ID NO: 47) (SEQ ID NO: 112) Ab80GTFSSYAIS GIIPIFGTANYAQKF ARGGGTFWSGSWA QG LY (SEQ ID NO: 5) (SEQ ID NO:27) (SEQ ID NO: 113) Ab81 GTFSSYAIS GIIPIFGTANYAQKF ARDSGNYDYWSGA QG LRY(SEQ ID NO: 5) (SEQ ID NO: 27) (SEQ ID NO: 114) Ab82 GSISSGGYYWSYIYYSGSTVYNPSLKS ARVSSSWYKA (SEQ ID NO: 13) (SEQ ID NO: 48) (SEQ ID NO:115) Ab83 GSFSGYYWS EIDHSGSTKYNPSLKS ARVGVVVGRPGYSA FDI (SEQ ID NO: 24)(SEQ ID NO: 49) (SEQ ID NO: 116) Ab84 YTFTSYGIS WISTYNGNTNYAQKARGSGSGYDSWYD LQG (SEQ ID NO: 12) (SEQ ID NO: 45) (SEQ ID NO: 99) Ab85FTFSSYGMH VIWYDGSNKYYADS AKDLGGYYGGAAY VKG GMDV (SEQ ID NO: 16) (SEQ IDNO: 41) (SEQ ID NO: 117) Ab86 FTFSSYGMH VISYDGSNKYYADS AKDGVYYGLGNWF VKGDP (SEQ ID NO: 16) (SEQ ID NO: 39) (SEQ ID NO: 118) Ab87 GSISSYYWSSIYYSGSTNYNPSLKS ARHGWDRVGWFDP (SEQ ID NO: 15) (SEQ ID NO: 38) (SEQ IDNO: 119) Antibody Name CDR L1 CDR L2 CDR L3 Ab1  RASQSVSSNLA GASTRATQQLPYWPPT (SEQ ID NO: 120) (SEQ ID NO: 138) (SEQ ID NO: 153) Ab2 RASQSVGSNLA GASTRAT QQYFFYPPT (SEQ ID NO: 121) (SEQ ID NO: 138) (SEQ IDNO: 154) Ab3  QASQDISNYLN DASNLAT QQPFNFPYT (SEQ ID NO: 122) (SEQ ID NO:139) (SEQ ID NO: 155) Ab4  RASQSVSSNLA SASTRAT QQDHDYPFT (SEQ ID NO:120) (SEQ ID NO: 140) (SEQ ID NO: 156) Ab5  RASQSVSSSYLA GASSRATQQYFSSPFT (SEQ ID NO: 123) (SEQ ID NO: 141) (SEQ ID NO: 157) Ab6 RASQSVSSYLA DASKRAT QQRVNLPPT (SEQ ID NO: 124) (SEQ ID NO: 142) (SEQ IDNO: 158) Ab7  RASQSVSSYLA DASKRAT QQRISYPIT (SEQ ID NO: 124) (SEQ ID NO:142) (SEQ ID NO: 159) Ab8  RASQSISSYLN GASSLQS QQIDDTPIT (SEQ ID NO:125) (SEQ ID NO: 143) (SEQ ID NO: 160) Ab9  RASQSVSSYLA DASNRATQQFSYWPWT (SEQ ID NO: 124) (SEQ ID NO: 144) (SEQ ID NO: 161) Ab10RASQSVSSSYLA GASSRAT QQHDSSPPT (SEQ ID NO: 123) (SEQ ID NO: 141) (SEQ IDNO: 162) Ab11 RASQSVSSDYLA GASSRAT QQDYSYPWT (SEQ ID NO: 126) (SEQ IDNO: 141) (SEQ ID NO: 163) Ab12 RASQSISSYLN AASSLQS QQEYAVPYT (SEQ ID NO:125) (SEQ ID NO: 145) (SEQ ID NO: 164) Ab13 RASQSVSSYLA DASNRATQQVSNYPIT (SEQ ID NO: 124) (SEQ ID NO: 144) (SEQ ID NO: 165) Ab14QASQDISNYLN DASNLET QQVDNIPPT (SEQ ID NO: 122) (SEQ ID NO: 146) (SEQ IDNO: 166) Ab15 QASQDISNYLN DASNLET QQFDTYPT (SEQ ID NO: 122) (SEQ ID NO:146) (SEQ ID NO: 167) Ab16 QASQDISNYLN DASNLET QQFLNFPT (SEQ ID NO: 122)(SEQ ID NO: 146) (SEQ ID NO: 168) Ab17 QASQDISNYLN DASNLET QQFFNFPT (SEQID NO: 122) (SEQ ID NO: 146) (SEQ ID NO: 169) Ab18 QASQDISNYLN DASNLETQQFIDLPFT (SEQ ID NO: 122) (SEQ ID NO: 146) (SEQ ID NO: 170) Ab19QASQDISNYLN DASNLET QQYYDLPFT (SEQ ID NO: 122) (SEQ ID NO: 146) (SEQ IDNO: 171) Ab20 RASQSVSSDYLA GASSRAT QQFSSHPFT (SEQ ID NO: 126) (SEQ IDNO: 141) (SEQ ID NO: 172) Ab22 RASQSVSSSYLA GASSRAT QQDDRSPYT (SEQ IDNO: 123) (SEQ ID NO: 141) (SEQ ID NO: 173) Ab23 KSSQSVLYSSNN WASTRESQQAYLPPIT KNYLA (SEQ ID NO: 127) (SEQ ID NO: 147) (SEQ ID NO: 174) Ab24RASQSISSYLN AASSLQS QQAFSPPPWT (SEQ ID NO: 125) (SEQ ID NO: 145) (SEQ IDNO: 175) Ab25 RASQSVSSSYLA GASSRAT QQDDRSPT (SEQ ID NO: 123) (SEQ ID NO:141) (SEQ ID NO: 176) Ab26 RASQSVSSYLA DASNRAT QQEFDLPFT (SEQ ID NO:124) (SEQ ID NO: 144) (SEQ ID NO: 177) Ab27 RASQSVSSYLA DASNRATQQYNNFPPT (SEQ ID NO: 124) (SEQ ID NO: 144) (SEQ ID NO: 178) Ab28RASQSVSSYLA DASKRAT QQRYLRPIT (SEQ ID NO: 124) (SEQ ID NO: 142) (SEQ IDNO: 179) Ab29 RASQSVSSSYLA GASSRAT QQPGAVPT (SEQ ID NO: 123) (SEQ ID NO:141) (SEQ ID NO: 180) Ab30 RASQSISSYLN GASSLQS QQVYITPIT (SEQ ID NO:125) (SEQ ID NO: 143) (SEQ ID NO: 181) Ab31 QASQDISNFLN DASNLETQQPVDLPFT (SEQ ID NO: 128) (SEQ ID NO: 146) (SEQ ID NO: 182) Ab32RASQSVSSYLA DASNRAT QQYSFFPPT (SEQ ID NO: 124) (SEQ ID NO: 144) (SEQ IDNO: 183) Ab33 RASQSVSSYLA DASNRAT QQDSSFPPT (SEQ ID NO: 124) (SEQ ID NO:144) (SEQ ID NO: 184) Ab34 RASQSISSYLN AASSLQS QQSDFPPWT (SEQ ID NO:125) (SEQ ID NO: 145) (SEQ ID NO: 185) Ab35 RASQSISSYLN AASSLQSQQGYSAPIT (SEQ ID NO: 125) (SEQ ID NO: 145) (SEQ ID NO: 186) Ab36RASQSVSSYLA DASNRAT QQLFDWPT (SEQ ID NO: 124) (SEQ ID NO: 144) (SEQ IDNO: 187) Ab37 RASQSVSSYLA DASNRAT QQRAFLFT (SEQ ID NO: 124) (SEQ ID NO:144) (SEQ ID NO: 188) Ab38 RASQSVSSYLA DASNRAT QQIDFLPYT (SEQ ID NO:124) (SEQ ID NO: 144) (SEQ ID NO: 189) Ab39 RASQSISSYLN AASSLQSQQVYSPPIT (SEQ ID NO: 125) (SEQ ID NO: 145) (SEQ ID NO: 190) Ab40RASQSISSYLN AASSLQS QQGYAAPIT (SEQ ID NO: 125) (SEQ ID NO: 145) (SEQ IDNO: 191) Ab41 RASQSVSSYLA DASNRAT QQRYALPIT (SEQ ID NO: 124) (SEQ ID NO:144) (SEQ ID NO: 192) Ab42 RASQSVSSSYLA GASSRAT QQYASPPIT (SEQ ID NO:123) (SEQ ID NO: 141) (SEQ ID NO: 193) Ab43 RASQSISRYLN AASSLQSQQVYSTPIT (SEQ ID NO: 129) (SEQ ID NO: 145) (SEQ ID NO: 194) Ab44RASQSVSSYLA DSSNRAT QQLVHWPT (SEQ ID NO: 124) (SEQ ID NO: 148) (SEQ IDNO: 195) Ab45 RASQSVSSNLA GASTRAT QQLDDWFT (SEQ ID NO: 120) (SEQ ID NO:138) (SEQ ID NO: 196) Ab46 RASQSVSSYLA DSSNRAT QQRSNYPIT (SEQ ID NO:124) (SEQ ID NO: 148) (SEQ ID NO: 197) Ab47 RASQSVSSYLA DASNRATQQRILYPIT (SEQ ID NO: 124) (SEQ ID NO: 144) (SEQ ID NO: 198) Ab48RASQSVSSYLA DASNRAT QQRAAYPIT (SEQ ID NO: 124) (SEQ ID NO: 144) (SEQ IDNO: 199) Ab49 RASQSVSSYLA DASKRAT QQRTSHPIT (SEQ ID NO: 124) (SEQ ID NO:142) (SEQ ID NO: 200) Ab50 RASQSVSSSYLA GASSRAT QQYAGSPFT (SEQ ID NO:123) (SEQ ID NO: 141) (SEQ ID NO: 201) Ab51 RASQSISSYLN AASSLQS QQFDDVFT(SEQ ID NO: 125) (SEQ ID NO: 145) (SEQ ID NO: 202) Ab53 RASQSVSSSYLAGASSRAT QQYVNSPFT (SEQ ID NO: 123) (SEQ ID NO: 141) (SEQ ID NO: 203)Ab54 RASQSINSYLN AASSLQS QQSDDDPFT (SEQ ID NO: 130) (SEQ ID NO: 145)(SEQ ID NO: 204) Ab55 RASQSVSSYLA DASNRAT QQLSTYPLT (SEQ ID NO: 124)(SEQ ID NO: 144) (SEQ ID NO: 205) Ab56 RASQSVSSYLA DASNRAT QQRSVYPIT(SEQ ID NO: 124) (SEQ ID NO: 144) (SEQ ID NO: 206) Ab57 RASQSVSSYLADASKRAT QQVSLFPLT (SEQ ID NO: 124) (SEQ ID NO: 142) (SEQ ID NO: 207)Ab58 RASQSISSWLA DASSLES LDYNSYSPIT (SEQ ID NO: 131) (SEQ ID NO: 149)(SEQ ID NO: 208) Ab59 QASQDISNYLN DASNLET QQHIALPFT (SEQ ID NO: 122)(SEQ ID NO: 146) (SEQ ID NO: 209) Ab60 RASQSVSSYLA DASKRAT QQRASMPIT(SEQ ID NO: 124) (SEQ ID NO: 142) (SEQ ID NO: 210) Ab61 RASQSVSSYLADSSNRAT QQAFNRPPT (SEQ ID NO: 124) (SEQ ID NO: 148) (SEQ ID NO: 211)Ab62 RASQSVSSYLA DASKRAT QQSSVHPYT (SEQ ID NO: 124) (SEQ ID NO: 142)(SEQ ID NO: 212) Ab63 RASQGIDSWLA AASSLQS QQAYSLPPT (SEQ ID NO: 132)(SEQ ID NO: 145) (SEQ ID NO: 213) Ab64 RASQSVSSNLA GASTRAT QQDDDGYT (SEQID NO: 120) (SEQ ID NO: 138) (SEQ ID NO: 214) Ab65 RASQSVSSYLA DASNRATQQDYSWPYT (SEQ ID NO: 124) (SEQ ID NO: 144) (SEQ ID NO: 215) Ab66RASQSVSSYLA DASNRAT QQRSAYPIT (SEQ ID NO: 124) (SEQ ID NO: 144) (SEQ IDNO: 216) Ab67 RASQSVSSYLA DASNRAT QQRSHFPIT (SEQ ID NO: 124) (SEQ ID NO:144) (SEQ ID NO: 217) Ab68 RASQSVSSYLA DASNRAT QQRANYPIT (SEQ ID NO:124) (SEQ ID NO: 144) (SEQ ID NO: 218) Ab69 RASQSVSSYLA DASNRATQQRADYPIT (SEQ ID NO: 124) (SEQ ID NO: 144) (SEQ ID NO: 219) Ab70RASQSVSSYLA DASNRAT QQRSVYPIT (SEQ ID NO: 124) (SEQ ID NO: 144) (SEQ IDNO: 206) Ab71 RASQSVSSSYLA GASNRAT QQAGSHPFT (SEQ ID NO: 123) (SEQ IDNO: 150) (SEQ ID NO: 220) Ab72 QASQDITNYLN DASNLET QQDVNYPPT (SEQ ID NO:133) (SEQ ID NO: 146) (SEQ ID NO: 221) Ab73 RASQSVSSNLA GASTRATQQDDNYPYT (SEQ ID NO: 120) (SEQ ID NO: 138) (SEQ ID NO: 222) Ab74RASQSVSSYLA DASNRAT QQRSTFPIT (SEQ ID NO: 124) (SEQ ID NO: 144) (SEQ IDNO: 223) Ab75 RASQSVSSYLA DASNRAT QQVSNYPFT (SEQ ID NO: 124) (SEQ ID NO:144) (SEQ ID NO: 224) Ab76 KSSQSVLYSSNN WASTRES QQYHDAPIT KNYLA (SEQ IDNO: 127) (SEQ ID NO: 147) (SEQ ID NO: 225) Ab77 KSSQSVLYSSNN WASTRESQQAYVVPPT KNYLA (SEQ ID NO: 127) (SEQ ID NO: 147) (SEQ ID NO: 226) Ab78RASQSVSSYLA DASNRAT QQADNWPFT (SEQ ID NO: 124) (SEQ ID NO: 144) (SEQ IDNO: 227) Ab79 RSSQSLLHSNGY LGSHRAS MQALESPRT NYLD (SEQ ID NO: 134) (SEQID NO: 151) (SEQ ID NO: 228) Ab80 RASQSVSSYLA DASNRAT QQYVNWPFT (SEQ IDNO: 124) (SEQ ID NO: 144) (SEQ ID NO: 229) Ab81 RASQSVSSYLA DASNRATQQSSNWPWT (SEQ ID NO: 124) (SEQ ID NO: 144) (SEQ ID NO: 230) Ab82RASQGISSWLA AASSLQS QQASTFPIT (SEQ ID NO: 135) (SEQ ID NO: 145) (SEQ IDNO: 231) Ab83 RASQGISSWLA AASSLQS QQRNSLPLT (SEQ ID NO: 135) (SEQ ID NO:145) (SEQ ID NO: 232) Ab84 RASQSISSYLN AASSLQS QQSYDFPIT (SEQ ID NO:125) (SEQ ID NO: 145) (SEQ ID NO: 233) Ab85 RASQDISSWLA AASSLQSQQEVDYPPLT (SEQ ID NO: 136) (SEQ ID NO: 145) (SEQ ID NO: 234) Ab86RASQSISSWLA KASSLES QQLNSYSPT (SEQ ID NO: 131) (SEQ ID NO: 152) (SEQ IDNO: 235) Ab87 RASQSVSRYLA DASNRAT QQYIFWPPT (SEQ ID NO: 137) (SEQ ID NO:144) (SEQ ID NO: 236)

Characterization of Ab1, Ab9, Ab14, Ab22, Ab45, and Ab65 Binding

Initial characterization of TREM2 antibodies involved determining theirability to bind TREM2 expressed on dendritic and other primary human ormouse immune cells. Cells were harvested, plated at 10⁵/ml in a 96 wellplate, washed, and incubated in 100 ul PBS containing 10-50 ug/ml Maband Fc blocking reagent for 1 hour in ice. Cells were then washed twiceand incubated in 100 ul PBS containing 5 ug/ml PE-conjugated secondaryantibody for 30 minutes in ice. Cells were washed twice in cold PBS andacquired on a BD FACS Canto. Data analysis and calculation of MFI valueswas performed with FlowJo (TreeStar) software version 10.0.7.

Antibodies Ab1, Ab9, Ab14, Ab22, Ab45, and Ab65 demonstrated binding toa mouse cell line (BWZ T2) expressing recombinant mouse TREM2, asindicated by positive TREM2 antibody staining detected via FACS analysis(black outlined histograms) (FIG. 21A). The negative isotype control(antibody Ab88) did not demonstrate binding. Antibodies Ab1, Ab9, Ab14,Ab22, Ab45, and Ab65 demonstrated antibody binding to WT (Trem+/+) bonemarrow derived mouse macrophages (BMMac, mMac), but not to TREM2deficient (TREM2−/−) mouse macrophages (BMMac, mMacs) (FIG. 21B).Antibodies Ab1, Ab9, Ab14, Ab22, Ab45, and Ab65 demonstrated binding toboth a human cell line (293) expressing recombinant Human TREM2 (FIG.22A) and to primary human dendritic cells (hDC) (FIG. 22B). Conversely,antibodies Ab43 and Ab60 bound to a human cell line expressingrecombinant human TREM2 (FIG. 22A), but did not bind to primary humandendritic cells (FIG. 22B).

Mean fluorescent intensities (MFI) values for cell types bound by TREM2antibodies Ab1, Ab9, Ab14, Ab22, Ab45, and Ab65 are listed in Table 9.Binding is compared to the parental mouse cell line (mTREM2 cell lineBWZ parental), primary human cell line (hTREM2 Parental Cell line(293)), primary mouse macrophages deficient in TREM2 (mMacs KO MFI), andprimary mouse dendritic cells deficient in TREM2 (mDC KO MFI). Resultsin Table 9 indicate that Ab1, Ab9, Ab14, Ab22, Ab45, and Ab65 bindspecifically to cell lines overexpressing human and mouse TREM2 on thecell membrane, but not to control cell lines that do not express TREM2.The antibodies also bind to primary human macrophages and primary mousemacrophages and dendritic cells. Binding to mouse primary cells isspecific, as it is not detected on primary cells derived from TREM2 KOmice.

TABLE 9 TREM2 Antibody Binding to Human and Mouse Cells mTrem2 hTrem2Cell line mTrem2 Parental hTrem2 (BWZ- Cell line Cell line Cell lineparental) (BWZ T2) (293) (293) mMacs mMacs mDC mDC hDC % Antibody MFIMFI MFI MFI KO MFI WT MFI KO MFI WT MFI positive Ab1  960 12626 86 149169.8 82.3 235 393 64.9 Ab9  1044 15691 83 2126 97.2 171.0 200 538 65.8Ab14 852 11550 87 1656 77.9 145.0 218 529 75.8 Ab22 828 12451 82 183767.2 110 0 191 451 76.9 Ab45 1022 16288 141 2058 86.2 141.0 277 652 78.8Ab65 1354 16122 93 1734 92.5 165.0 260 642 76.9

The binding affinity of each anti-TREM2 antibody was determined bymeasuring their K_(D) by ForteBio or MSD-SET. ForteBio affinitymeasurements were performed as previously described (Estep et al, (2013)MAbs 5(2):270-8). Briefly, ForteBio affinity measurements were performedby loading IgGs on-line onto AHQ sensors. Sensors were equilibratedoff-line in assay buffer for 30 min and then monitored on-line for 60seconds for baseline establishment. Sensors with loaded IgGs wereexposed to 100 nM antigen for 5 minutes, then transferred to assaybuffer for 5 min for off-rate measurement. Kinetics were analyzed usingthe 1:1 binding model.

Equilibrium affinity measurements were performed as previously described(Estep et al, (2013) MAbs 5(2):270-8). Solution equilibrium titrations(SET) were performed in PBS+0.1% IgG-Free BSA (PBSF) with antigen heldconstant at 50 pM and incubated with 3- to 5-fold serial dilutions ofantibody starting at 10 nM. Antibodies (20 nM in PBS) were coated ontostandard bind MSD-ECL plates overnight at 4° C. or at room temperaturefor 30 min. Plates were then blocked for 30 min with shaking at 700 rpm,followed by three washes with wash buffer (PBSF+0.05% Tween 20). SETsamples were applied and incubated on the plates for 150s with shakingat 700 rpm followed by one wash. Antigen captured on a plate wasdetected with 250 ng/mL sulfotag-labeled streptavidin in PBSF byincubation on the plate for 3 min. The plates were washed three timeswith wash buffer and then read on the MSD Sector Imager 2400 instrumentusing 1× Read Buffer T with surfactant. The percent free antigen wasplotted as a function of titrated antibody in Prism and fit to aquadratic equation to extract the K_(D). To improve throughput, liquidhandling robots were used throughout MSD-SET experiments, including SETsample preparation.

Table 10 lists values representing the binding affinity (K_(D)) ofantibodies Ab1, Ab9, Ab14, Ab22, Ab45, and Ab65 to a human TREM2 Fcfusion protein (hTREM2-Fc), a human monomeric His tagged TREM2 protein(hTREM2-HIS), and a mouse TREM2 Fc fusion protein (mTREM2-Fc).

TABLE 10 Binding affinity of TREM2 antibodies IgG K_(D) hTREM2-Fc IgGK_(D) hTREM2- IgG K_(D) mTREM2- Antibody (M) Avid HIS (M) Monovalent Fc(M) Avid Ab1 7.05E−10 6.67E−09 4.86E−09 Ab9 3.48E−10 6.32E−09 N.B. Ab145.51E−10 3.19E−09 6.20E−10 Ab22 3.06E−10 1.01E−09 3.40E−10 Ab45 2.29E−106.54E−10 N.B. Ab65 5.46E−10 5.01E−09 1.56E−09

Example 41: TREM2 Antibodies Induce the Expression of CD83 and CD86 onHuman Dendritic Cells (DCs) and Induce T Cell Proliferation

To evaluate the ability of anti-TREM2 antibodies to modify expression ofCD83 and CD86, both plate bound and soluble antibodies were incubatedwith dendritic cells (DCs), and the expression of CD83, CD86, CCR7, andphosphorylated ERK were measured. To evaluate the ability of anti-TREM2antibodies to modulate T cell proliferation, DCs were incubated with Tcells and anti-TREM2 antibodies, and the level of T cell proliferationwas measured.

Antibodies were plated overnight at 4C in 12 well plates at 2 or 5 ug/mlin PBS. Wells were washed 3× with PBS the next day. On day 5, immaturehuman DCs were harvested and plated at 1 million cells per well andincubated at 37C, 5% CO₂ in the absence of cytokine. FACS analysis ofCD86, CD83, CD11c, HLA-DR, and LIN (BD Biosciences) was performed on aBD FACS Canto 48 hours later. Data analysis was performed with FlowJo(TreeStar) software version 10.0.7. Levels of CD83, CD86, and CCR7 wereevaluated for CD11c+HLA-DR+LIN− cell populations. For intracellular ERKphosphorylation, cells were fixed with 1% formaldehyde, permeabilizedwith cytofix/cytoperm kit (BD), and intracellular Erk phopshorylationwas determined with flow cytometry after staining with PE-ERK antibody(BD).

Alternatively, Day 5 immature human dendritic cells were plated at100,000 cells per well in a U-bottom non-TC treated 96 well plate inmedia without cytokine. Antibodies were added at 5 ug/ml with or withoutLPS-removed anti-human secondary (Jackson ImmunoResearch) at 20 ug/ml.FACS analysis for CD86, CD83, CD11c, HLA-DR, and LIN (BD Biosciences)was performed 48 hrs post antibody addition as previously described.

Additionally, Day 5 immature dendritic cells (CD14⁻CD1 c⁺ LIN⁻) wereplated in 12 well dishes coated the previous day with 2 ug/ml antibody.Plates were washed 3 times with PBS before addition of T cells. CD4⁺ Tcells from non-autologous donors were isolated and labeled with CFSEbefore addition to DCs in ratio of 1:10, 1:50, or 1:250. CD3/CD28 Dynalbeads serve as a positive control. Day 5 post co-culture cells wereanalyzed by flow cytometry on a BD FACSCanto II for CFSE dilution.Percent CFSE^(hi) compared to CFSE^(lo) cells were calculated for eachcondition with FlowJo (TreeStar).

Plate bound TREM2 antibodies Ab1, Ab9, Ab14, Ab22, Ab45, and Ab65increased the frequency of CD83+CD86+ DCs compared to the isotypecontrol antibody Ab88 (FIG. 23A). Soluble antibodies Ab1, Ab9, Ab14,Ab45, and Ab65, when cross-linked with anti-human secondary antibody,induced expression of CD86 on DCs. Conversely, cross-linked antibodyAb22 and non-cross-linked soluble antibodies did not induce CD86expression (FIG. 23B). Based on these results, TREM2 antibodies Ab1,Ab9, Ab14, Ab22, Ab45, and Ab65 function as agonists to induce theexpression of inflammatory surface markers CD83 and CD86 on humandendritic cells.

Example 42: TREM2 Antibodies Ab1, Ab9, Ab14, Ab20, Ab22, Ab45, and Ab65Induce Syk Phosphorylation

Spleen tyrosine kinase (Syk) is an intracellular signaling molecule thatfunctions downstream of TREM2 by phosphorylating several substrates,thereby facilitating the formation of a signaling complex leading tocellular activation and inflammatory processes. The ability of agonistTREM2 antibodies to induce Syk activation was determined by culturinghuman and mouse macrophages and primary human dendritic cells andmeasuring the phosphorylation state of Syk protein in cell extracts.

Bone marrow-derived macrophages (BMDM), WT mouse BMDM, TREM2 knockout(KO) mouse BMDM, and primary human dendritic cells were starved for 4hours in 1% serum RPMI and then removed from tissue culture dishes withPBS-EDTA, washed with PBS, and counted. The cells were coated withfull-length agonist TREM2 antibodies Ab1, Ab9, Ab14, Ab20, Ab22, Ab45,Ab65, non-agonistic antibodies (Ab16, Ab77), or control antibodies(Ab89, or Ab92) for 15 minutes on ice. After washing with cold PBS,cells were incubated at 37° C. for the indicated period of time in thepresence of goat anti-human IgG. After stimulation, cells were lysedwith lysis buffer (1% v/v NP-40%, 50 Mm Tris-HCl (pH 8.0), 150 mM NaCl,1 mM EDTA, 1.5 mM MgCl₂, 10% glycerol, plus protease and phosphataseinhibitors) followed by centrifugation at 16,000 g for 10 min at 4° C.to remove insoluble materials. Lysates were then immunoprecipitated withanti-Syk Ab (N-19 for BMDM or 4D10 for human DCs, Santa CruzBiotechnology). Precipitated proteins were fractionated by SDS-PAGE,transferred to PVDF membranes and probed with anti-phosphotyrosine Ab(4G10, Millipore). To confirm that all substrates were adequatelyimmunoprecipitated, immunoblots were reprobed with anti-Syk Ab (Abcam,for BMDM) or anti-Syk (Novus Biological, for human DCs). Visualizationwas performed with the enhanced chemiluminescence (ECL) system (GEhealthcare), as described (e.g., Peng et al., (2010) Sci Signal.,3(122): ra38).

TREM2 antibodies Ab1, Ab9, Ab14, Ab20, Ab22, Ab45, and Ab65 inducedTREM-2 mediated Syk phosphorylation in BMDMs, (FIG. 24A). Sykphosphorylation induced by Antibodies Ab1, Ab9, and Ab45 is TREM-2specific, as Syk phosphorylation is not induced when TREM2 KO BMDM areused as a control (FIG. 24B). TREM2 antibodies Ab22, Ab45, and Ab65induce Syk phopshorylation in primary human dendritic cells (FIG. 24C).Non agonistic antibodies (Ab16 and Ab77) or control antibodies (Ab89 andAb92) did not induce Syk phosphorylation. Based on these results, TREM2antibodies Ab1, Ab9, Ab14, Ab20, Ab22, Ab45, Ab65 function as agoniststo induce Syk phosphorylation in macrophages and dendritic cells.

Example 43: TREM2 Antibodies Ab1, Ab9, Ab14, Ab22, Ab45, and Ab65Compete with TREM2 Ligand for Binding to Human and Mouse TREM2

The ability of agonist TREM2 antibodies to recognize the ligand-bindingsite on TREM2 was evaluated through competitive binding assays with E.coli cells expressing a putative TREM2 ligand.

E. coli were grown in 10 ml LB media O/N, harvested by centrifuging, andwashed twice in 10 ml PBS. E. coli were then heat-inactivated byincubating in a 70° C. water bath for 30 min. E. coli were labeled withCellTracker DeepRed (ThermoFisher/Invitrogen, 1 uM final concentration)and subsequently washed thrice in 10 ml PBS and resuspended in 1 ml PBSat a concentration of 10⁸/ml. For competition binding, bacteria wereadded to a mouse TREM2 and DAP12 expressing cell line (BWZ), or to a BWcell line expressing a human TREM2/DAP12 fusion protein, together with10 μg/ml of full-length agonist TREM2 antibodies and incubated for onehour on ice. Cells were analyzed via FACS for binding of CellTrackerlabeled bacteria to the cell lines.

TREM2 antibodies Ab1, Ab9, Ab14, Ab22, Ab45, and Ab65 inhibited thebinding of E. coli bacteria to both human and mouse cells, indicatingcompetitive binding of the antibodies to the ligand-binding site onTREM2 (FIG. 25). Non-agonistic control TREM2 antibodies (Ab66 and Ab68)partially inhibited bacterial binding to human cells expressing TREM2,but did not inhibit binding to mouse TREM2, indicating that they areweaker competitors of ligand binding.

Example 44: TREM2 Antibodies Ab1, Ab9, Ab22, Ab45, and Ab65 Induce DAP12Phosphorylation in Mouse Macrophages

TREM2 signals through DAP12, leading downstream to activation of PI3Kand other intracellular signals. The ability of agonist TREM2 antibodiesto induce DAP12 activation was determined by culturing mouse macrophagesand measuring the phosphorylation state of DAP12 protein in cellextracts.

Before stimulation with mAbs, mouse wild-type (WT) bone marrow-derivedmacrophages (BMDM) and TREM2 knockout (KO) BMDM were starved for 4h in1% serum RPMI. 15×10⁶ cells were incubated in ice for 15 min withfull-length agonistic or control antibodies. Cells were washed andincubated at 37° C. for the indicated period of time in the presence ofgoat anti-human IgG. After stimulation, cells were lysed with lysisbuffer (1% v/v NP-40%, 50 Mm Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA,1.5 mM MgCl₂, 10% glycerol, plus protease and phosphatase inhibitors),followed by centrifugation at 16,000 g for 10 min at 4° C. to removeinsoluble materials. Cell lysate was immunoprecipitated with a secondTREM2 antibody (R&D Systems). Precipitated proteins were fractionated bySDS-PAGE, transferred to PVDF membranes, and probed withanti-phosphotyrosine Ab (4G10, Millipore). The membrane was stripped andreprobed with anti-DAP12 antibody (Cells Signaling, D7G1X). Each celllysate used for TREM2 immunoprecipitations contained an equal amount ofproteins, as indicated by a control Ab (anti-actin, Santa Cruz).

DAP12 co-precipitated with TREM2 and was phosphorylated in WTmacrophages incubated with agonist TREM2 antibodies Ab1, Ab9, Ab22,Ab45, and Ab65 (FIGS. 26A and 26B). Conversely, no DAP12 phosphorylationwas observed in TREM2 KO (TREM2^(−/−)) macrophages incubated withantibodies Ab1, Ab9, Ab22, or Ab45 (FIG. 26B). These results demonstratethat agonistic antibodies Ab1, Ab9, Ab22, and Ab45 inducephosphorylation of TREM-2-associated DAP12 in a TREM-2 specific manner,as DAP12 phosphorylation is absent in TREM-2 deficient BMDM.

Example 45: Epitope Mapping of TREM2 Antibodies and Comparison of TREM2Antibodies to Reference TREM2 Antibodies

TREM2 antibodies were tested for their ability to bind 15 or 25 merpeptides spanning the entire human and mouse TREM2. The TREM2 antibodieswere also compared to a reference TREM2 antibody by determining theirTREM2 binding region.

Linear 15-mer peptides were synthesized based on the sequence of humanor mouse TREM2, with a 14 residue overlap. In addition, linear 25-merpeptides were synthesized based on sequence of human or mouse TREM2 witha 1 residue shift. The binding of TREM2 antibodies to each of thesynthesized peptides was tested in an ELISA based method. In this assay,the peptide arrays were incubated with primary antibody solution(overnight at 4° C.). After washing, the peptide arrays were incubatedwith a 1/1000 dilution of an antibody peroxidase conjugate (SBA, cat.nr. 2010-05) for one hour at 25° C. After washing, the peroxidasesubstrate 2,2′-azino-di-3-ethylbenzthiazoline sulfonate (ABTS) and 2μl/ml of 3% H2O2 were added. After one hour, the color development wasmeasured. The color development was quantified with a charge coupleddevice (CCD) camera and an image processing system.

To evaluate antibody binding regions, human TREM2-Fc was incubated withimmobilized full-length agonist TREM2 antibodies Ab1, Ab9, Ab14, Ab22,Ab45, Ab63, Ab65, or Ab87, and TREM2 antibody MAB17291 (R&D Systems) wassubsequently added. Epitope binning of the antibodies was performed on aForte Bio Octet Red384 system (Pall Forte Bio Corporation, Menlo Park,Calif.) using a standard sandwich format binning assay (see Estep et al,(2013) MAbs 5(2):270-8). Control anti-target IgG was loaded onto AHQsensors and unoccupied Fc-binding sites on the sensor were blocked witha non-relevant human IgG1 antibody. The sensors were then exposed to 100nM target antigen followed by a second anti-target antibody. Data wasprocessed using ForteBio's Data Analysis Software 7.0. Additionalbinding by the second antibody after antigen association indicates anunoccupied epitope (non-competitor), while no binding indicates epitopeblocking (competitor).

Antibodies Ab1, Ab9, Ab28, and Ab29 yielded strong and robust bindingexclusively for peptides from Set1 and Set2, which are derived from thesequence of human TREM2. All four antibodies bound peptides that containthe region between amino acid residues 139-146 of human TREM2(¹³⁹GDLWFPGE¹⁴⁶ (SEQ ID NO: 237)) (FIG. 27A). The epitope regionrecognized by Ab1, Ab9, Ab28, and Ab29 corresponds to amino acidresidues 139-146 of SEQ ID NO: 1 and has the amino acid sequence of:GDLWFPGE (SEQ ID NO: 237).

Antibodies Ab45 and Ab65 bound only to 25-mer peptides from Set2 andSet4. Both antibodies recognized a highly conserved region of TREM2 nearits C-terminus between amino acid residues 140-153 of the human TREM2(¹⁴⁰DLWFPGESESFEDA¹⁵³ (SEQ ID NO: 238)) and mouse TREM2(¹⁴⁰DLWVPEESSSFEGA¹⁵³ (SEQ ID NO: 239)) (FIG. 27B). The epitope regionrecognized by Ab45 and Ab65 corresponds to amino acid residues 140-153of SEQ ID NO: 1 and has the amino acid sequence of: DLWFPGESESFEDA (SEQID NO: 238).

The epitope region recognized by reference antibody MAB17291 was alsodetermined. Reference antibody MAB17291 was found to recognize a firstpeptide that contains the region between amino acid residues 130-144 ofhuman TREM2 (¹³⁰ADPLDHRDAGDLWFP¹⁴⁴ (SEQ ID NO: 240)), and a secondpeptide that contains the region between amino acid residues 158-170 ofhuman TREM2 (¹⁵⁸ SISRSLLEGEIPF¹⁷⁰ (SEQ ID NO: 241)). The epitope regionsrecognized by MAB17291 correspond to amino acid residues 130-144 and158-170 of SEQ ID NO: 1 and have the amino acid sequences of:ADPLDHRDAGDLWFP (SEQ ID NO: 240) and SISRSLLEGEIPF (SEQ ID NO: 241).

Reference antibody MAB17291 was able to simultaneously bind TREM2 withantibody Ab1, Ab9, Ab14, Ab22, Ab45, or Ab65, but not with antibody Ab63or Ab87 (FIG. 28). These results demonstrate that agonist TREM2antibodies Ab1, Ab9, Ab14, Ab22, Ab45, and Ab65 bind different regionsof the TREM2 protein than does reference antibody MAB17291.

Example 46: Summary of TREM2 Antibody Functional Studies

Table 11 summarizes results of the functional studies described inExamples 41-45 above. Antibodies Ab1, Ab9, Ab14, Ab22, Ab45, and Ab65demonstrated induction of CD83 and CD86 on human dendritic cells (hDC),with higher induction observed with plate-bound antibody compared tocross-linked soluble antibody (values in table represent percentage ofCD83+CD86+ cells). Antibodies Ab1, Ab9, Ab14, Ab22, Ab45, and Ab65induced variable levels of Syk phosphorylation in human dendritic cells(hDC), mouse dendritic cells (mDC), and mouse macrophages (mMac), andwere able to mimic ligand binding (blocking bacterial binding) to humanand mouse TREM2.

TABLE 11 TREM2 Antibody Functional Studies Induction Induction Induce ofof Phospho CD86 & CD86 & TREM2 Blocking CD83 CD83 Induction Inductionand Bacterial Blocking Human hDC Phospho Phospho DAP12 binding BacterialAntibody hDC plate solution Syk hDC Syk mDC mMac hTREM2 hTREM2 Ab1  87.23.0 −/+ ++ ++ +++ +++ Ab9  79.3 5.6 −/+ +++ ++++ +++ +++ Ab14 78.7 1.9 +++ N/D +++ +++ Ab22 74.5 1.2 ++ ++ ++ +++ +++ Ab45 65.5 4.3 +++ +++ ++++++ +++ Ab65 74 1.7 ++ +++ +++ +++ +++ Isotype − − − − − − − control

Example 47: Analysis of Tumor Growth in TREM2 Deficient Mice

Groups of 10 TREM2 wild-type (WT), TREM2 heterozygous (HET), and TREM2knock-out (KO) mice (sex and age-matched littermates, 8 weeks old (+/−2weeks)) are challenged subcutaneously with tumor cells (for example1×10⁵-1×10⁶ MC38 colon carcinoma, Lewis Lung carcinoma, or B16 melanomacells) suspended in 100 ul PBS. Animals are anesthetized with isofluraneprior to implant. Tumor growth is monitored with a caliper biweekly tomeasure tumor growth starting at day 4. The endpoint of the experimentis a tumor volume of 2000 mm³ or 60 days. Tumor growth and % survivalare the outcome measures. Reduced tumor take and growth rate and areduced number of tumor infiltrating immune suppressor macrophagesindicate increased effector T cell influx into the tumor in TREM2 KOmice.

To determine the number of infiltrating tumor associated immunesuppressor macrophages and T cells, groups of 6-8 sex and age-matchedlittermates are used. 8 week old (+/−2 weeks) WT-HET-KO littemates arechallenged subcutaneously with tumor cells (e.g. 1×10⁵-1×10⁶ MC38, LewisLung, or 16 cells) suspended in 200 ul Matrigel (Matrigel Matrix GrowthFactor Reduced; BD). Animals are anesthetized with isoflurane prior toimplant. 7 and 10 days after tumor injection, the matrigel plug isresected, incubated for 1 hour at 37° C. with 1 mg/ml Collagenase D(Sigma), dissociated to obtain a single-cell suspension, and filteredthrough a cell strainer. To determine the amount of T cells recruited inthe tumor and the ratio between effector T cells and regulatory T cells,5×10⁶ cells are stained with anti-CD45.2 PercpCy5.5, anti-CD3-FITC,anti-CD8-PECY7, anti-CD4-APC, anti-FoxP3-PE (BD), and DAPI. To determinethe amount of monocyte/macrophage lineage cells recruited into thetumor, 5×10⁶ cells are stained with anti-CD45.2 PercpCy5.5,anti-CD11b-PECY7, anti-F4/80-FITC, anti-Ly6C/G-APC, anti-CD86-PE, andDAPI. Cells are acquired on a BD FACS Canto. Data analysis is performedwith FlowJo (TreeStar) software version 10.0.7.

Example 48: Analysis of the Anti-Cancer Effect of TREM2 AntagonisticAntibodies

Groups of 10 C57B16/NTac mice at 8 weeks (+/−2 weeks) of age arechallenged subcutaneously with tumor cells (e.g. 1×10 to 1×10⁶ MC38,Lewis Lung, or B16 cells) suspended in 100 ul PBS. Animals areanesthetized with isoflurane prior to implant. Starting at day 2, groupsof mice are injected i.p. every 3 days for 4 doses with 200 ug of eachof anatagonisitc anti-TREM2 antibodies, such as those described inExamples 38 and 40. Tumor growth is monitored with a caliper biweekly tomeasure tumor growth starting at day 4. The endpoint of the experimentis a tumor volume of 2000 mm³ or 60 days. Tumor growth and % survivalare the outcome measures. Reduced tumor take and growth rate, reducednumber of tumor infiltrating immune suppressor macrophages, andincreased effector T cell influx into the tumor indicate the anti-cancereffects of blocking anti-TREM2 antibodies.

Example 49: Analysis of Additive Anti-Tumor Effect of CombinationTherapy that Combines TREM2 Antibodies with Antibodies AgainstInhibitory Checkpoint Proteins or Inhibitory Cytokines/Chemokines andtheir Receptors

Groups of 15 C57B16/NTac mice at 8 weeks (+/−2 weeks) of age arechallenged subcutaneously with tumor cells as described in Example 35.Animals are anesthetized with isoflurane prior to implant. Starting atday 2, mice are injected i.p. every 3 days for 4 doses with 200 uganti-TREM2 antibodies alone or in combination with antibodies againstcheckpoint proteins (e.g. anti-PDL1 mAb clone 10F.9G2 and/or anti-CTLA4mAb clone UC10-4F10-11) at day 3, 6, and 9. Treatment groups includeanti-TREM2; anti-CTLA4; anti-PDL1; anti-TREM2+anti-CTLA4;anti-TREM2+anti-PDL1; and isotype control. Tumor growth is monitoredwith a caliper biweekly to measure tumor growth starting at day 4. Theendpoint of the experiment is a tumor volume of 2000 mm³ or 60 days.Tumor growth and % survival are the outcome measures. A decrease intumor growth and an increase in % survival with combination therapyindicate that anti-TREM2 antibodies have additive or synergistictherapeutic effects with anti-checkpoint antibodies. Antagonisticantibodies against checkpoint molecules include antibodies against PDL1,PDL2, PD1, CTLA4, B7-H3, B7-H4, HVEM, BTLA, KIR, GAL9, TIM3, A2AR,LAG-3, and Phosphatidyl Serine. Antagonist antibodies against inhibitorycytokines include antibodies against CCL2, CSF-1, and IL-2.

Example 50: Analysis of Additive Anti-Tumor Effect of CombinationTherapy that Combines TREM2 Antibodies with Antibodies that ActivateStimulatory Checkpoint Proteins

Groups of 15 C57B16/NTac mice at 8 weeks (+/−2 weeks) of age arechallenged subcutaneously with tumor cells as described in Example 35.Animals are anesthetized with isoflurane prior to implant. Starting atday 2, mice are injected i.p. every 3 days for 4 doses with 200 uganti-TREM2 antibodies alone or in combination with agonistic antibodiesthat activate stimulatory checkpoint proteins (e.g. OX40 or ICOS mAb) atday 3, 6, and 9. Tumor growth is monitored with a caliper biweekly tomeasure tumor growth starting at day 4. The endpoint of the experimentis a tumor volume of 2000 mm³ or 60 days. Tumor growth and % survivalare the outcome measures. A decrease in tumor growth and an increase in% survival with combination therapy indicate that anti-TREM2 antibodieshave additive or synergistic therapeutic effects with stimulatorycheckpoint antibodies. Stimulatory checkpoint antibodies includeagonistic/stimulatory antibodies against CD28, ICOS, CD137, CD27, CD40,and GITR.

Example 51: Analysis of Additive Anti-Tumor Effect of CombinationTherapy that Combines TREM2 Antibodies with Stimulatory Cytokines

Groups of 15 C57B16/NTac mice at 8 weeks (+/−2 weeks) of age arechallenged subcutaneously with tumor cells as described in Example 35.Animals are anesthetized with isoflurane prior to implant. Starting atday 2, mice are injected i.p. every 3 days for 4 doses with 200 uganti-TREM2 antibodies alone or in combination with stimulatory cytokines(e.g. IL-12, IFN-a). Tumor growth is monitored with a caliper biweeklyto measure tumor growth starting at day 4. The endpoint of theexperiment is a tumor volume of 2000 mm³ or 60 days. Tumor growth and %survival are the outcome measures. A decrease in tumor growth and anincrease in % survival with combination therapy indicate that anti-TREM2antibodies have additive or synergistic therapeutic effects withimmune-stimulatory cytokines. Stimulatory cytokines include IFN-a/b,IL-2, IL-12, IL-18, GM-CSF, and G-CSF.

Example 52: Analysis of Ability of TREM2 Antibody Fabs to StimulateViability of Innate Immune Cells

The agonistic functionality of plate bound, cross-linked anti-TREM2antibody Fab fragments derived from antibodies Ab22, Ab45, and Ab65 wasevaluated in innate immune cells (e.g., macrophages).

Wild-type (WT) and TREM2 knock-out (KO) mouse bone marrow derivedmacrophages were cultured in the presence of M-CSF and plate bound TREM2antibody Fabs, and cell viability was measured.

Macrophages isolated from the bone marrow of WT and KO mice were platedon non-tissue-culture-treated 96-well plates, pre-coated with either12.5 nM or 100 nM of cross-linked Ab22, Ab45, or Ab65 Fabs. Cells werecultured for 48 hours in the presence of 10 ng/ml M-CSF. Analysis ofviability was performed using Cell Titer Glo kit (Promcga). Plates wereread with a BioTek Synergy Microplate Reader using GEN5 2.04 software.

Cross-linked TREM2 Fab fragments derived from antibodies Ab22, Ab45, andAb65 increased the number of viable mouse bone marrow-derivedmacrophages compared to isotype control Fab Ab88, as indicated by ahigher % increased survival (FIG. 29). This enhancement in cellviability was not observed in KO mouse macrophages, with the exceptionof Ab65. These data indicate that the biological activity of Ab22 andAb45 is TREM2 specific, and that plate bound, cross-linked Ab22 and Ab45Fab fragments function as agonists to increase the survival ofmacrophages cultured in M-CSF.

Example 53: Analysis of Ability of TREM2 Antibodies to Decrease Survivalof Innate Immune Cells

The antagonistic functionality of both soluble, non-cross-linkedanti-TREM2 antibody Fab fragments derived from antibodies and soluble,full-length anti-TREM2 antibodies was evaluated in innate immune cells(e.g., macrophages).

Wild-type (WT) and TREM2 knock-out (KO) mouse bone marrow derivedmacrophages were cultured in the presence of M-CSF and soluble TREM2antibody Fabs or soluble full-length antibodies, and cell viability wasmeasured.

Macrophages isolated from the bone marrow of WT and KO mice were platedon non-tissue-culture-treated 96-well plates in the presence of 20 ng/mlM-CSF and increasing amounts of the indicated soluble, non-cross-linkedTREM2 antibody Fabs derived from antibodies Ab22, Ab45, and Ab65, orsoluble, full-length antibodies Ab9, Ab14, Ab22, Ab45, and Ab65. Eachcondition was plated in triplicate. Analysis of viability was performedusing Cell Titer Glo kit (Promega) 3 days later. Plates were read with aBioTek Synergy Microplate Reader using GEN5 2.04 software.

In FIGS. 30A and 30B, the “NT” dotted line indicates the average cellviability obtained with untreated macrophages (no antibody added). The“no MCSF” dotted line indicates the average cell viability obtained whenmacrophages were cultured in the absence of M-CSF.

When macrophage cell viability was evaluated with soluble,non-cross-linked TREM2 antibody Fabs, the results indicated thatsoluble, non-cross-linked TREM2 Fab fragments derived from antibody Ab45decreased cell viability (FIG. 30A). In contrast, soluble,non-cross-linked TREM2 Fab fragments derived from antibody Ab22 did notinhibit viability and had an effect comparable to the isotype controlAb99 (FIG. 30A). Soluble, non-cross-linked TREM2 Fab fragments derivedfrom antibody Ab65 partially inhibited cell viability (FIG. 30A). Theresults demonstrate that the soluble, non-cross-linked Fab derived fromTREM2 antibodies Ab45 and Ab65 can function as antagonists and inhibitthe survival of macrophages in vitro.

When macrophage cell viability was evaluated with soluble, full-lengthantibodies, antibodies Ab14, Ab45, Ab65, and Ab9 decreased cellviability (FIG. 30B). Soluble, full-length antibody Ab22 had an effecton cell viability that was nearly comparable to that of isotype controlAb91 (FIG. 30B). The results demonstrate that soluble, full-length TREM2antibodies Ab14, Ab45, Ab65, and Ab9 can function as antagonists, whennot cross-linked or clustered, and inhibit the survival of macrophages.

The results of these experiments indicate that TREM2 antibodies Ab45,Ab65, and Ab9, in the absence of clustering, can inhibit the survival ofinnate immune cells such as macrophages. In contrast, TREM2 antibodyAb22, even in the absence of clustering, does not inhibit the survivalof innate immune cells such as macrophages.

Example 54: Analysis of the Ability of TREM2 Antibodies to InduceTREM2-Dependent Genes

The ability of plate bound full-length anti-TREM2 antibodies Ab1, Ab9,Ab10, Ab14, Ab15, Ab17, Ab18, Ab20, Ab22, Ab24, Ab25, Ab28, Ab29, Ab45,Ab54, Ab51, Ab64, Ab65, and Ab66 to activate TREM2-dependent genes wasevaluate using a luciferase reporter gene under the control of an NFAT(nuclear factor of activated T-cells) promoter.

A cell line derived from mouse thymus lymphoma T lymphocytes BW5147.G.1.4 (ATCC® TIB48™) was infected with mouse Trem2 and Dap12, and withCignal Lenti NFAT-Luciferase virus (Qiagen). Full-length anti-TREM2antibodies were plate bound at 10 ug/ml in DPBS on tissue-culturetreated clear bottom white 96 well plates (100 ul/well), overnight at 4°C. Wells were rinsed thrice with DPBS and subsequently were plated at100,000 cells/well in media with 1% serum. As a positive control forsignaling, PMA (0.05 ug/ml) and ionomycin (0.25 uM) were added together.Cells were incubated for 6 hours and luciferase activity was measured byadding OneGlo Reagent (Promega) to each well and incubating 3 min at RTon a plate shaker. Luciferase signal was measured using a BioTek platereader.

As shown in FIG. 31A and FIG. 31B, anti-TREM2 antibodies Ab1, Ab9, Ab10,Ab14, Ab15, Ab17, Ab20, Ab22, Ab45, Ab54, Ab64, Ab65, and Ab66 increasedluciferase activity, indicating that the antibodies were able to induceTREM2-dependent gene transcription. As shown in FIG. 31C, plate boundphosphatidylserine (PS) induces TREM2-dependent gene expression. It isbelieved that PS is a natural ligand of TREM2. Thus, the results inFIGS. 31A-C indicate that agonist anti-TREM2 antibodies can mimic anatural ligand of TREM2.

Example 55: Analysis of the Ability of TREM2 Antibodies to InhibitTREM2-Dependent Genes

The ability of soluble, full-length anti-TREM2 antibodies Ab9, Ab14,Ab22, Ab45, and Ab65 to inhibit TREM2-dependent genes was evaluate usinga luciferase reporter gene under the control of an NFAT (nuclear factorof activated T-cells) promoter.

A cell line derived from mouse thymus lymphoma T lymphocytes BW5147.G.1.4 (ATCC® TIB48™) was infected with mouse Trem2 and Dap12, and withCignal Lenti NFAT-Luciferase virus (Qiagen). Soluble, full-lengthanti-TREM2 antibodies were added at increasing concentration to thecells. Cells were incubated for 6 hours at 37° C. and luciferaseactivity was measured using OneGlo Reagent (Promega).

The cells display tonic TREM2-dependent signaling due to either thepresence of an endogenous ligand or to spontaneous receptor aggregation,which leads to TREM2 signaling.

The dotted line in FIG. 32 indicates the levels of TREM2 activitywithout stimulation.

As shown in FIG. 32, soluble, full-length anti-TREM2 antibodies Ab9,Ab14, Ab45, and Ab65 were able to inhibit tonic, TREM2-dependent geneexpression. In contrast, soluble, full-length anti-TREM2 antibody Ab22did not appear to block tonic TREM2 signaling (FIG. 32).

Example 56: Summary of TREM2 Antibody Agonistic and AntagonisticActivity

Table 12 summarizes results of the functional studies described inExamples 52-55 above. Antibodies Ab1, Ab9, Ab14, Ab22, Ab45, and Ab65demonstrated agonistic activity in activating TREM2-dependent geneexpression using either a luciferase reporter gene (FIG. 31A) or abeta-GAL reporter gene (Table 12). As indicated in Table 12, Ab1 andAb65 showed an increased level of gene induction, compared to Ab9, Ab22,and Ab45 when the beta-GAL reporter gene was used (Table 12). AntibodiesAb22, Ab45, and Ab65 also demonstrated agonistic activity in stimulatingcell survival of innate immune cells (FIG. 29). Table 12 furthersummarizes results demonstrating the antagonistic effects of soluble,non-cross-linked antibodies Ab9, Ab14, Ab45, and Ab65 in inhibiting cellsurvival of innate immune cells (FIG. 30). In contrast, soluble,non-cross-linked antibody Ab22 had minimal antagonistic activity ininhibiting cell survival (FIG. 30).

TABLE 12 TREM2 Antibody Functional Studies Luciferase Luciferase betaGALSurvival Antagonistic antibody Agonistic Agonistic Agonistic activity inantibody antibody antibody solution/antagonistic Antibody activityactivity activity format Ab1 ++++ +++ ND ND Ab9 ++++ + ND ++++ Ab14 ++++ND ND +++ Ab22 ++++ + +++ − Ab45 ++ + ++ ++++ Ab65 +++ +++ + +++ Isotype− − − − control

Example 57: Summary of Binding and Functional Studies for Further TREM2Antibodies

Table 13 summarizes the results of binding and functional studies forfurther full-length TREM2 antibodies generated as described in Example40. The results below were obtained using the methods described inExamples 40 and 42.

TABLE 13 Binding and functional studies for other TREM2 Antibodies IgGK_(D) mTrem2 hTrem2 IgG K_(D) IgG K_(D) mTREM2- Cell Cell hTREM2-FchTREM2- Fc (M) binding binding Phospho Phospho Antibody (M) Avid HIS (M)t Avid MFI MFI Syk hDC Syk mDC Ab2  5.92E−10 2.41E−08 N.B. 1432 1407 NDND Ab3  4.90E−10 1.42E−08 N.B. 488 1693 ND ND Ab4  9.71E−10 P.F. N.B.295 1343 ND ND Ab5  6.97E−10 3.00E−08 N.B. 310 1336 − ND Ab6  4.44E−09P.F. 8.51E−09 228 816 ND ND Ab7  1.05E−09 3.34E−08 1.11E−09 10934 1249ND ND Ab8  7.23E−10 1.50E−08 N.B. 523 1623 ND ND Ab10 5.44E−10 4.79E−09P.F. 11486 1788 ++ ++ Ab11 9.34E−10 7.20E−09 1.19E−09 5495 1909 ND NDAb12 1.19E−09 2.68E−09 7.91E−09 1552 1930 − ND Ab13 1.49E−09 6.67E−081.71E−09 3246 971 ND ND Ab15 6.05E−10 3.03E−09 2.80E−09 4755 1611 ND +Ab16 1.09E−09 2.47E−09 1.36E−09 6382 1545 ND − Ab17 7.92E−10 3.46E−091.67E−09 10312 1604 ND + Ab18 5.13E−10 6.77E−09 N.B. 5164 1924 ++ + Ab194.65E−10 4.49E−09 N.B. 2304 1993 ++ ND Ab20 6.58E−10 3.62E−09 N.B. 112451556 ND + Ab23 9.67E−10 P.F. 6.83E−09 919 2653 ND ND Ab24 4.79E−10 P.F.9.32E−09 2760 1882 ND + Ab25 6.84E−10 4.51E−09 5.98E−09 2497 1884 ND −Ab26 4.81E−10 3.60E−09 N.B. 2551 1421 ND ND Ab27 1.33E−09 P.F. 6.39E−092163 1316 ND ND Ab28 9.15E−10 P.F. 7.45E−09 5248 1487 ND − Ab29 2.36E−09P.F. 3.27E−09 4518 1193 ND − Ab30 1.32E−09 P.F. N.B. 399 1421 ND ND Ab314.51E−10 6.82E−09 N.B. 551 1760 ND ND Ab32 9.95E−10 2.48E−08 1.35E−08756 1202 ND ND Ab33 1.42E−09 1.60E−08 N.B. 413 1352 ND ND Ab34 7.06E−102.70E−08 N.B. 395 1770 ND ND Ab35 2.21E−10 7.82E−09 6.44E−09 931 1391 NDND Ab36 4.65E−10 4.01E−09 N.B. 1258 1896 ND ND Ab37 1.58E−09 P.F. N.B.1433 1434 ND ND Ab38 9.60E−10 1.31E−08 N.B. 297 1832 ND ND Ab39 2.13E−09P.F. 6.41E−09 588 9029 ND ND Ab40 1.97E−10 7.97E−09 6.03E−09 577 1452 NDND Ab41 2.14E−09 P.F. 8.64E−09 373 1017 ND ND Ab42 4.03E−09 P.F. N.B.387 1234 ND ND Ab43 4.37E−09 P.F. N.B. 418 918 ND ND Ab44 5.62E−109.46E−09 N.B. 1206 1249 ND ND Ab46 4.45E−10 1.68E−08 2.46E−09 4257 1528ND ND Ab47 4.31E−10 7.37E−09 1.45E−09 10123 1833 ND ND Ab48 3.50E−109.08E−09 1.41E−09 9770 1412 ND ND Ab49 4.35E−10 1.74E−08 2.08E−09 43151392 ND ND Ab50 7.29E−10 3.64E−08 N.B. 325 1435 ND ND Ab51 2.84E−101.64E−09 N.B. 12077 1928 ++ + Ab52 1.51E−09 5.75E−09 8.96E−11 15613 1411+++ +++ Ab53 6.35E−10 2.16E−08 N.B. 843 1338 ND ND Ab54 5.00E−103.02E−09 N.B. 11001 1931 ND ++ Ab55 2.22E−09 9.17E−08 1.17E−09 6221 911ND ND Ab56 1.03E−09 5.18E−08 2.71E−09 743 1157 ND ND Ab57 4.36E−101.83E−08 5.98E−10 10226 1227 ND ND Ab58 5.75E−10 2.73E−09 N.B. 1870 1698ND ND Ab59 8.54E−10 5.72E−09 N.B. 3901 1754 ND ND Ab60 5.59E−09 P.F.P.F. 1871 741 ND ND Ab61 4.61E−09 P.F. N.B. 10517 1101 ND ND Ab627.72E−10 1.42E−08 P.F. 4746 1647 ND ND Ab63 1.13E−09 4.96E−08 N.B. 17221347 ND ND Ab64 9.45E−10 6.57E−08 P.F. 8676 1458 +++ + Ab66 1.98E−096.07E−08 3.88E−09 4098 1161 ND + Ab67 6.60E−10 3.45E−08 2.92E−09 62071118 ND ND Ab68 6.68E−09 1.09E−07 3.26E−09 2103 1135 ND ND Ab69 3.75E−095.97E−08 3.40E−09 629 957 ND ND Ab70 6.26E−09 P.F. 3.45E−09 3265 833 NDND Ab71 2.77E−09 6.47E−08 N.B. 804 1556 ND ND Ab72 8.39E−10 2.94E−08P.F. 3746 1500 ND ND Ab73 4.10E−09 P.F. N.B. 1117 1307 ND ND Ab749.43E−09 P.F. 4.91E−09 1360 645 ND ND Ab75 5.88E−09 P.F. N.B. 276 676 NDND Ab76 4.84E−09 P.F. N.B. 1604 580 ND ND Ab77 P.F. P.F. N.B. 1650 951ND ND Ab78 8.51E−10 3.73E−08 N.B. 433 1435 ND ND Ab79 1.07E−08 P.F. N.B.738 900 ND ND Ab80 5.72E−09 P.F. 8.85E−09 593 897 ND ND Ab81 1.01E−08P.F. N.B. 400 994 ND ND Ab82 1.10E−08 N.B. 5.82E−09 712 371 ND ND Ab838.21E−09 N.B. N.B. 1576 684 ND ND Ab84 1.16E−09 6.04E−08 N.B. 799 1148ND ND Ab85 5.72E−09 P.F. 2.19E−09 1121 460 ND ND Ab86 3.89E−09 P.F.6.55E−09 355 840 ND ND Ab87 9.51E−09 N.B. 4.48E−09 1228 414 ND ND 88control No Binding ND ND 90.7 187 − − 89 control No Binding ND ND 142185 − − Luciferase Agonistic Antibody antibody activity Ab10 ++++ Ab15++++ Ab17 ++++ Ab18 − Ab20 +++ Ab24 − Ab25 − Ab28 − Ab51 +++ Ab29 − Ab54+++ Ab64 +++ Ab66 +++ Ab89 − Ab90 − Isotype Control − PMA/Ion +++

Example 58: Analysis of Anti-Stroke Effect of Agonistic TREM2 Antibodies

Transient occlusion of the middle cerebral artery (MCAO)—a model thatclosely resembles human stroke is used to induce cerebral infarction inmice. Monofilament (70SPRe, Doccol Corp, USA) is introduced into theinternal carotid artery through an incision of the right common carotidartery. The middle cerebral artery is occluded for 30 minutes with arange of reperfusion times (6 h, 12 h, 24 h, 2 d, 7 d and 28 d). Theeffect of surgery is controlled using sham animals at 12 h and at 7 d.Sham animals undergo the same surgical procedure without occlusion ofthe middle cerebral artery. MCAO animals treated with agonisticanti-TREM2 antibodies or control antibodies are tested for infarctvolumetry, acute inflammatory response (12 h reperfusion), transcriptionof pro-inflammatory cytokines TNFa, IL-1a, and IL-1b, microglialactivity (CD68, Iba1), transcription of chemokines CCL2 (MCP1), CCL3(MIP1a and the chemokine receptor CX3CR1 and invasion of CD3-positiveT-cells (Sieber et al. (2013) PLoS ONE 8(1): e52982.doi:10.1371/journal.pone.0052982.).

Example 59: Analysis of the Protective Effect of Antagonist TREM2Antibodies in Respiratory Tract Infections

To evaluate the ability of antagonist TREM2 antibodies to delay,prevent, or treat bacterial respiratory tract infections, a preclinicalmouse model involving challenge of C57B16 mice with Streptococcuspneumoniae is used. This model involves intranasal (i.n.) administrationof 105 CFU S. pneumnoniae serotype 3 (ATCC 6303) as described (see,e.g., Sharif O et al, 2014 PLoS Pathog. 2014 June; 10(6): e1004167; andSchabbauer G et al, 2010 J Immunol 185: 468-476). In this model ˜90% WTC57B16 mice succumb to infection within 6 days post infection.

Ten to fifteen mice/group are challenged with S. pneumoniae andconcomitantly are treated with antagonist anti-TREM2 antibodies everyother day starting from day 0. The first dose of anti-TREM2 antibodiesis administered 3 hours prior to challenge with S. pneumonia. Mice aremonitored daily for 15 days to check for death events. % of micesurviving bacteria challenge is determined.

In separate experiments, count of bacterial load and cytokine expressionin the blood and in the lungs is also determined. 24 or 48 hours afterinfection blood is collected in EDTA-containing tubes and plated on agarplates to enumerate bacterial CFU in the plasma. Plasma is stored at−20° C. for cytokine analysis by ELISA. Lungs are harvested, homogenizedand plated on agar plates to enumerate bacterial CFU, or incubated for30 min in lysis buffer and supernatants analyzed for cytokinemeasurements.

In separate experiments, lungs are collected 40 hours post bacterialinfection, fixed in 10% formalin, and embedded in paraffin for H&Epathology analysis.

Example 60: Analysis of the Protective Effect of Antagonist TREM2Antibodies in Sepsis

To evaluate the ability of antagonist TREM2 antibodies to delay,prevent, or treat sepsis, a preclinical mouse model involving systemicchallenge of C57B16 mice with LPS is used. This model involvesintraperitoneal (i.p.) administration of 37 mg/ml LPS as described (see,e.g., Gawish R et al, 2014 FASEB J). In this model >95% WT C57B16 micesuccumb infection within 40 hours post LPS injection.

Cohorts of mice are challenged with LPS and concomitantly are treatedwith antagonist anti-TREM2 antibodies every day starting from day 0. Thefirst dose of anti-TREM2 antibodies is administered 3 hours prior tochallenge with LPS. Mice are monitored every ˜4 hours during daytime, tocheck for death events. Percentage of mice surviving LPS challenge isdetermined.

In separate experiments, peritoneal lavage fluid (PLF) is collected.Supernatants are stored at −20° C. for cytokine analysis by ELISA;pelleted cells are counted to quantify inflammatory cells recruited inthe peritoneal cavity. Similar studies can be conducted to test theefficacy of TREM2 antibodies in other models of infection (see, e.g.,Sun et al., (2013) Invest Ophthalmol Vis Sci. 17; 54(5):3451-62).

Example 61: Analysis of the Protective Effect of Antagonist TREM2Antibodies in Acute and Chronic Colitis

To evaluate the ability of antagonist anti-TREM2 antibodies to delay,prevent, or treat colitis, preclinical mouse models of acute or chroniccolitis are used. For DSS-induced colitis, mice receive 3% DSS indrinking water ad libiturn for 8 days. For TNBS-induced colitis, miceare anesthetized and treated with an intra-rectal injection of 3 mg TNBSin 20% ethanol (vol/vol) or vehicle alone as a control. For the chroniccolitis model, all mice are treated with 3 cycles of 2% DSS for 5 days,followed by a 10-day recovery period. For all models, weight loss, stoolconsistency, and presence of fecal occult blood are monitored daily andused to calculate the disease activity index, as described (see, e.g.,Correale C, 2013, Gastroenterology, February 2013, pp. 346-356.e3).

Cohorts of mice are treated with antagonist anti-TREM2 antibodies everyday starting from day 0 and subjected to DSS or TNBS administration.Mice are monitored every day, to check for weight loss, stoolconsistency, and presence of fecal occult blood were monitored daily andused to calculate the disease activity index, as described (see, e.g.,S. Vetrano, Gastroenterology, 135 (2008), pp. 173-184).

In separate experiments, endoscopic and histological images of mucosaldamage are collected to evaluate inflammatory cell infiltration andmucosal damage. Similar studies can be conducted to test the benefit ofTREM2 antibodies in other models of autoimmunity including Crohn'sdisease, inflammatory bowel disease, and ulcerative colitis (see, e.g.,Low et al., (2013) Drug Des Devel Ther.; 7: 1341-1357; and Sollid etal., (2008) PLoS Med 5(9): e198).

Example 62: Analysis of the Protective Effect of Agonist TREM2Antibodies in Wound Healing

To evaluate the ability of agonistic anti-TREM2 antibodies to increasecolonic wound repair following injury, a mouse model of biopsy injury inthe colon is used. In this model, the endoscope with outer operatingsheath is inserted to the mid-descending colon and the mucosa issurveyed to the ano-rectal junction. Then, a single full thickness areaof the entire mucosa and submucosa is removed with flexible biopsyforceps with a diameter of 3 French, avoiding penetration of themuscularis propria. Each mouse is biopsy injured at 3-5 sites along thedorsal side of the colon (see, e.g., Seno H, 2008, Proc Natl Acad SciUSA. 2009 Jan. 6; 106(1): 256-261).

Cohorts of mice are treated with agonist anti-TREM2 antibodies 2 or 3days after biopsy injury. Mice are monitored every day for 15 days, tocheck for weight loss and wound healing by measuring the surface area oflesions.

Example 63: Analysis of the Protective Effect of Antagonist TREM2Antibodies in Retinal Degeneration

Antagonist TREM2 antibodies decrease the accumulation and/or function ofinflammatory macrophages, and as a result delay, prevent and/or treatage-related macular degeneration (AMD).

AMD is a degenerative disease of the outer retina. It is thought thatinflammation, particularly inflammatory cytokines and macrophages,contribute to AMD disease progression.

The presence of macrophages in the proximity of AMD lesions isdocumented, in the drusen, Bruch's membrane, choroid and retina.Macrophages release tissue factor (TF) and vascular endothelial growthfactor (VEGF), which triggers the expansion of new blood vesselsformation in patients showing choroidal neovasulcarization.

The type of macrophage present in the macular choroid changes with age,displaying elevated levels of M2 macrophages in older eyes compared toyounger eyes. However, advanced AMD maculae had higher M1 to M2 rationscompared to normal autopsied eyes of similar age. (see, e.g., Cao X etal, (2011), Pathol Int 61(9): pp528-35). This suggests a link betweenclassical M1 macrophage activation in the eye in the late onset of AMDprogression.

Retinal microglia cells are tissue-resident macrophages that are alsonormally present in the inner retina. In the event of damage, microgliacan be activated and act as mediator of inflammation. Activatedmicroglia has been detected in the AMD tissue samples and has beenproposed as one potential contributor of inflammatory processed thatlead to AMD pathogenesis (Gupta et al., (2003) Exp Eye Res.,76(4):463-71.). The ability of antagonist TREM2 antibodies to prevent,delay, or reverse AMD is tested in one or more of AMD models (see, e.g.,Pennesi et al., (2012) Mol Aspects Med.; 33(4): 487-509).

Overall inflammatory macrophages (either M1 and/or activated microglia)are documented to correlate with AMD disease progression and thereforerepresent a therapeutic target for antagonist TREM2 antibodies. Similartherapeutic benefit can be achieved in glaucoma and genetic forms orretinal degeneration such as retinitis pigmentosa.

The ability of antagonist TREM2 antibodies to prevent, delay, or reverseretinal ganglion cell degeneration in glaucoma is tested in a glaucomamodel (see, e.g., El-Danaf et al., (2015). J Neurosci. 11;35(6):2329-43). Likewise, the theraputic benefit of REM2 in geneticallyinduced retinal degeneration and retinitis pigmentosa is tested asdescribed in Chang et al., (2002) Vision Res.; 42(4):517-25, and in“Retinal Degeneration Rat Model Resource Availability of P23H andS334ter Mutant Rhodopsin Transgenic Rats and RCS Inbred and RCS CongenicStrains of Rats,” M M LaVail, Jun. 30, 2011.

Example 64: Analysis of the Protective Effect of Antagonist TREM2Antibodies in Adipogenesis and Diet-Induced Obesity

To test the effect of antagonist TREM2 antibodies in adipogenesis andobesity, a mouse model of high-fat diet (HFD) is used (see, e.g., Parket al., (2015) Diabetes. 64(1):117-27).

Example 65: Analysis of the Protective Effect of TREM2 Antibodies inMalaria

TREM2 expression in the nonparenchymal liver cells closely correlateswith resistance to liver stage infection with the maliaria agentPlasmodium berghei (Gonçalves et al., (2013) Proc Natl Acad Sci 26;110(48):19531-6). Without wishing to be bound to theory, it is believedthat TREM2 antibodies increase resistance to liver stage infection withP. berghei.

The ability of TREM2 antibodies to increase resistance to malariainfection is tested as described in Gonçalves et al., (2013) Proc NatlAcad Sci 26; 110(48):19531-6. Briefly, GFP-expressing P. berghei ANKAsporozoites are obtained by dissection of infected salivary glands fromAnopheles stephensi mosquitoes. Sporozoite suspensions in RPMI mediumare injected i.v. in 100 μL of inocula containing 10⁴ sporozoites permouse. Livers are collected at 40 h after injection or survival, andparasitemia is followed for 28 days. For experimental cerebral malariascoring, neurologic symptoms are monitored from day 5 after injection.

Example 66: Analysis of the Protective Effect of Antagonist TREM2Antibodies in Osteoporosis

Bone is a dynamic organ constantly remodeled to support calciumhomeostasis and structural needs. The osteoclast is the cell responsiblefor removing both the organic and inorganic components of bone. Theosteoclast is derived from hematopoietic progenitors in the macrophagelineage and differentiates in response to the tumor necrosis factorfamily cytokine receptor activators of NFκB ligand. Osteoclasts, theonly bone-resorbing cells, are central to the pathogenesis ofosteoporosis and osteopetrosis (Novack et al., (2008) Annual RevPathol., 3:457-84).

Osteoporosis is a progressive bone disease that is characterized by adecrease in bone mass and density which can lead to an increased risk offracture. It is mostly manifested in the first years followingmenopause, when bone turnover is accelerated, with increased activity ofboth osteoclasts and osteoblasts. Owing to an imbalance in the processesof resorption and synthesis, however, the net effect is bone loss, whichis largely trabecular. Thus, the most prevalent sites of fracture inosteoporosis are the wrist, femoral neck, and vertebral bodies, in whichthe trabecular structure is key to overall bone strength. Acceleratedosteoclast differentiation and increased bone resorption capacity,resulting in osteoporosis have been described in animal models lackingthe expression of TREM2 (Otero et al (2012) J. Immunol. 188, 2612-2621).

Reduced osteoclast function results in osteopetrosis, with increasedbone mass and elimination of bone marrow space, as observed in animalmodels lacking DAP12 ITAM signaling adapter and resulting in asignificant defect in differentiation of osteoclast-like cells (Koga, etal., (2004) Nature 428: 758-763).

Without wishing to be bound by theory, it is believed that administeringan antagonist anti-TREM2 antibody of the present disclosure can prevent,reduce the risk of, and/or treat osteoporosis. In some embodiments,administering an agonist anti-TREM2 antibody may induce one or moreTREM2 activities in an individual having osteopetrosis (e.g., DAP12phosphorylation, Syk activation, and accelerated differentiation intoosteoclasts) (Peng et al (2010). Sci Signal. 2010 18; 3 122; andHumphrey et al., (2006) J Bone Miner Res., 21(2):237-45).

Example 67: Ability of Ab22 and Ab45 TREM2 Antibodies to Block Inductionof TREM2-Dependent Gene Expression

Antibodies Ab22 and Ab45 were tested for their ability to blockinduction of TREM2-dependent gene expression by lipid lignads using aLuciferase reporter system.

Phosphatidylserine (PS, Avanti Lipids) and Sphingomiyelin (SM, AvantiLipids) were dissolved in methanol and plated onto Immulon 96 wellplates. Methanol was evaporated overnight at RT and cells were added thenext day. BWZ cells expressing mouse TREM2 and DAP12 in conjunction withan NFAT:luciferase reporter (Qiagen) were harvested and added at 100,000cells/well in DMEM+1% FBS. Cells were either plated alone or incombination with 50 or 5 nM human IgG. After six hours, luciferaseactivity was measured using OneGlo reagent (Promega) and a Biotek platereader, according to the manufacturer's instructions.

Ab45 blocked the induction of TREM2 dependent gene expression by both PSand SM, as indicated by a decrease in luminescence (FIG. 33).

Example 68: Analysis of the Ability of TREM2 Antibodies to InduceTREM2-Dependent Cytokine Genes

The ability of plate bound anti-TREM2 Fab antibodies Ab22 and Ab65 toactivate TREM2-dependent genes was evaluated using primary mousemacrophages.

Bone marrow-derived macrophages (BMDM) were generated with M-CSF for 5days. BMDM (10⁵/well) was seeded on 96 well plates that had beenpreviously coated with the Fab portion of the antibodies Ab22 and Ab65(50 nM). Ultrapure LPS (100 ng/ml Escherichia coli 0111:B4) was addedafter the plates were spun for 1 min at 1200 rpm. After 18 h ofstimulation with LPS, cytokines present in cell supernatants weremeasured with cytometric bead array (CBA; BD Biosciences, CBA mouseinflammation kit).

As shown in FIGS. 34A and 34B, plate bound anti-TREM2 Fab antibody Ab22increased the levels of IL-6 and MCP-1, indicating that the antibodieswere able to activate TREM2-dependent cytokine genes in primary immunecells.

1.-93. (canceled)
 94. A method of preventing, reducing risk, or treatingan individual having a disease, disorder, or injury selected from thegroup consisting of dementia, frontotemporal dementia, Alzheimer'sdisease, Nasu-Hakola disease, and multiple sclerosis, comprisingadministering to the individual a therapeutically effective amount of anisolated antibody that binds to a TREM2 protein, wherein the isolatedantibody induces one or more TREM2 activities, and wherein the isolatedantibody promotes survival of one or more innate immune cells orincreases expression of IL-6.
 95. (canceled)
 96. The method of claim 94,wherein the individual has a heterozygous variant of TREM2, wherein thevariant comprises one or more substitutions selected from the groupconsisting of: i. a glutamic acid to stop codon substitution in thenucleic acid sequence encoding amino acid residue Glu14 of SEQ ID NO: 1;ii. a glutamine to stop codon substitution in the nucleic acid sequenceencoding amino acid residue Gln33 of SEQ ID NO: 1; iii. a tryptophan tostop codon substitution in the nucleic acid sequence encoding amino acidresidue Trp44 of SEQ ID NO: 1; iv. an arginine to histidine amino acidsubstitution at an amino acid corresponding to amino acid residue Arg47of SEQ ID NO: 1; v. a tryptophan to stop codon substitution in thenucleic acid sequence encoding amino acid residue Trp78 of SEQ ID NO: 1;vi. a valine to glycine amino acid substitution at an amino acidcorresponding to amino acid residue Val126 of SEQ ID NO: 1; vii. anaspartic acid to glycine amino acid substitution at an amino acidcorresponding to amino acid residue Asp134 of SEQ ID NO: 1; and viii. alysine to asparagine amino acid substitution at an amino acidcorresponding to amino acid residue Lys186 of SEQ ID NO:
 1. 97. Themethod of claim 94, wherein the individual has a heterozygous variant ofTREM2, wherein the variant comprises a guanine nucleotide deletion at anucleotide corresponding to nucleotide residue G313 of the nucleic acidsequence encoding SEQ ID NO: 1; a guanine nucleotide deletion at anucleotide corresponding to nucleotide residue G267 of the nucleic acidsequence encoding SEQ ID NO: 1; or both.
 98. The method of claim 94,wherein the individual has a heterozygous variant of DAP12, wherein thevariant comprises one or more variants selected from the groupconsisting of: i. a methionine to threonine substitution at an aminoacid corresponding to amino acid residue Met1 of SEQ ID NO: 2; ii. aglycine to arginine amino acid substitution at an amino acidcorresponding to amino acid residue Gly49 of SEQ ID NO: 2; iii. adeletion within exons 1-4 of the nucleic acid sequence encoding SEQ IDNO: 2; iv. an insertion of 14 amino acid residues at exon 3 of thenucleic acid sequence encoding SEQ ID NO: 2; and v. a guanine nucleotidedeletion at a nucleotide corresponding to nucleotide residue G141 of thenucleic acid sequence encoding SEQ ID NO:
 2. 99.-102. (canceled)